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Infection of human immunodeficiency virus type 1 (HIV-1) is subject to restriction by cellular factors. Serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3) proteins represent two of these restriction factors, which inhibit HIV-1 entry into target cells. Both proteins impede fusion of the viral membrane with the cellular membrane and the formation of a viral fusion pore, and both are countered by the HIV-1 envelope glycoprotein (Env). Given the immense and lasting pressure which Env endures from host adaptive immune responses, it is important to understand whether and how HIV-1 Env is able to maintain the resistance to SERINC5 and IFITM3 throughout the course of infection. We have thus examined a panel of HIV-1 Env clones that were isolated at different stages of viral infection-transmission, acute, and chronic. While HIV-1 Env clones from the transmission stage are resistant to both SERINC5 and IFITM3, as infection progresses into the acute and chronic stages, the resistance to IFITM3 but not to SERINC5 is gradually lost. We further discovered a significant correlation between the resistance of HIV-1 Env to soluble CD4 inhibition and the resistance to SERINC5 but not to IFITM3. Interestingly, the miniprotein CD4 mimetic M48U1 sensitizes HIV-1 Env to the inhibition by SERINC5 but not IFITM3. Together, these data indicate that SERINC5 and IFITM3 exert differential inhibitory pressures on HIV-1 Env over different stages of HIV-1 infection and that HIV-1 Env uses varied strategies to resist these two restriction factors. IMPORTANCE HIV-1 Env protein is exposed to the inhibition not only by humoral response, but also by host restriction factors, including serine incorporator 5 (SERINC5) and interferon-inducible transmembrane 3 (IFITM3). This study investigates how HIV-1 envelope glycoprotein (Env) manages to overcome the pressures from all these different host inhibition mechanisms over the long course of viral infection. HIV-1 Env preserves the resistance to SERINC5 but becomes sensitive to IFITM3 when infection progresses into the chronic stage. Our study also supports the possibility of using CD4 mimetic compounds to sensitize HIV-1 Env to the inhibition by SERINC5 as a potential therapeutic strategy., (Copyright © 2020 American Society for Microbiology.)

One striking characteristic of certain herpesviruses is their ability to induce rapid and widespread RNA decay in order to gain access to host resources. This phenotype is induced by viral endoribonucleases, including SOX in Kaposi's sarcoma-associated herpesvirus (KSHV), muSOX in murine gammaherpesvirus 68 (MHV68), BGLF5 in Epstein-Barr virus (EBV), and vhs in herpes simplex virus 1 (HSV-1). Here, we performed comparative transcriptome sequencing (RNA-seq) upon expression of these herpesviral endonucleases in order to characterize their effect on the host transcriptome. Consistent with previous reports, we found that approximately two-thirds of transcripts were downregulated in cells expressing any of these viral endonucleases. Among the transcripts spared from degradation, we uncovered a cluster of transcripts that systematically escaped degradation from all tested endonucleases. Among these escapees, we identified C19ORF66 and reveal that this transcript is protected from degradation by its 3' untranslated region (UTR). We then show that C19ORF66 is a potent KSHV restriction factor by impeding early viral gene expression, suggesting that its ability to escape viral cleavage may be an important component of the host response to viral infection. Collectively, our comparative approach is a powerful tool to pinpoint key regulators of the viral-host interplay and led us to uncover a novel KSHV regulator. IMPORTANCE Viruses are master regulators of the host gene expression machinery. This is crucial to promote viral infection and to dampen host immune responses. Many viruses, including herpesviruses, express RNases that reduce host gene expression through widespread mRNA decay. However, it emerged that some mRNAs escape this fate, although it has been difficult to determine whether these escaping transcripts benefit viral infection or instead participate in an antiviral mechanism. To tackle this question, we compared the effect of the herpesviral RNases on the human transcriptome and identified a cluster of transcripts consistently escaping degradation from all tested endonucleases. Among the protected mRNAs, we identified the transcript C19ORF66 and showed that it restricts Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Collectively, these results provide a framework to explore how the control of RNA fate in the context of viral-induced widespread mRNA degradation may influence the outcome of viral infection., (Copyright © 2019 American Society for Microbiology.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens affecting many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. PRRSV infection can cleave mitochondrial antiviral signaling protein (MAVS) and inhibit the induction of type I interferon. The antiviral effector molecules that are involved in host protective responses to PRRSV infection are not fully understood. Here, by using transcriptome sequencing, we found that a zinc finger antiviral protein, ZAP, is upregulated in MAVS-transfected Marc-145 cells and that ZAP suppresses PRRSV infection at the early stage of replication. We also found that the viral protein Nsp9, an RNA-dependent RNA polymerase (RdRp), interacts with ZAP. The interacting locations were mapped to the zinc finger domain of ZAP and N-terminal amino acids 150 to 160 of Nsp9. These findings suggest that ZAP is an effective antiviral factor for suppressing PRRSV infection, and they shed light on virus-host interaction. IMPORTANCE PRRSV continues to adversely impact the global swine industry. It is important to understand the various antiviral factors against PRRSV infection. Here, a zinc finger protein, termed ZAP, was screened from MAVS-induced antiviral genes by transcriptome sequencing, and it was found to remarkably suppress PRRSV replication and interact with PRRSV Nsp9. The zinc finger domain of ZAP and amino acids 150 to 160 of Nsp9 are responsible for the interaction. These findings expand the antiviral spectrum of ZAP and provide a better understanding of ZAP antiviral mechanisms, as well as virus-host interactions., (Copyright © 2019 American Society for Microbiology.)

Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition. IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding-induced epitopes., (Copyright © 2017 American Society for Microbiology.)

Dengue virus (DENV) is a member of the genus Flavivirus and can cause severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is induced by DENV in an interferon-dependent manner, displays antiviral activity against DENV, and localizes to the DENV replication complex. IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 and Xrn1, localizes to the DENV replication complex and associates with DENV proteins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data serve to characterize an interferon-stimulated gene with antiviral activity against DENV, as well as to propose a mechanism of activity involving the processing of viral RNA. IMPORTANCE Dengue virus, a member of the family Flaviviridae , can result in a life-threatening illness and has a significant impact on global health. Dengue virus has been shown to be particularly sensitive to the effects of type I interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. A better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. Here, we examine the influence of the interferon-stimulated gene IRAV ( FLJ11286 ) on dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also interacts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection., (Copyright © 2017 Balinsky et al.)

Unlabelled: The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-α) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-γ. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells., Importance: Cytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation., Importance: The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity., Importance: dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to specific viral mRNAs and repressing mRNA expression. Here we report that ZAP inhibits expression of murine gammaherpesvirus 68 (MHV-68) M2, which plays important roles in establishment and maintenance of viral latency. Downregulation of endogenous ZAP in cells harboring latent MHV-68 promoted lytic replication of the virus. These results suggest that ZAP inhibits M2 expression and regulates the maintenance of MHV-68 latency.

The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.

In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

Reassortment is an important driving force for influenza virus evolution, and a better understanding of the factors that affect this process could improve our ability to respond to future influenza pandemics and epidemics. To identify factors that restrict the generation of reassortant viruses, we cotransfected human embryonic kidney cells with plasmids for the synthesis of viral RNAs of both A/equine/Prague/1/56 (Prague; H7N7) and A/Yokohama/2017/03 (Yokohama; H3N2) viruses together with the supporting protein expression plasmids. Of the possible 256 genotypes, we identified 29 genotypes in 120 randomly plaque-picked reassortants examined. Analyses of these reassortants suggested that the formation of functional ribonucleoprotein (RNP) complexes was a restricting factor, a finding that correlated with the activities of RNP complexes composed of different combinations of the proteins from the two viruses, as measured in a minigenome assay. For at least one nonfunctional RNP complex (i.e., Prague PB2, Prague PB1, Yokohama PA, and Prague NP), the lack of activity was due to the inability of the three polymerase subunit proteins to form a heterotrimer. Adaptation of viruses possessing a gene encoding a chimera of the PA proteins of the two viruses and the remaining genes from Prague virus resulted in compensatory mutations in the PB2 and/or PA protein. These results indicate substantial incompatibility among the gene products of the two test viruses, a critical role for the RNP complex in the generation of reassortant viruses, and a functional interaction of PB2 and PA.

Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia DeltaE3L mutant virus triggers the production of beta interferon (IFN-beta), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. DeltaE3L induction of IFN-beta, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with DeltaE3L, an event that depends on MAVS. The response of keratinocytes to DeltaE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.

Early during the infection process, rotavirus causes the shutoff of cell protein synthesis, with the nonstructural viral protein NSP3 playing a vital role in the phenomenon. In this work, we have found that the translation initiation factor 2alpha (eIF2alpha) in infected cells becomes phosphorylated early after virus infection and remains in this state throughout the virus replication cycle, leading to a further inhibition of cell protein synthesis. Under these restrictive conditions, however, the viral proteins and some cellular proteins are efficiently translated. The phosphorylation of eIF2alpha was shown to depend on the synthesis of three viral proteins, VP2, NSP2, and NSP5, since in cells in which the expression of any of these three proteins was knocked down by RNA interference, the translation factor was not phosphorylated. The modification of this factor is, however, not needed for the replication of the virus, since mutant cells that produce a nonphosphorylatable eIF2alpha sustained virus replication as efficiently as wild-type cells. In uninfected cells, the phosphorylation of eIF2alpha induces the formation of stress granules, aggregates of stalled translation complexes that prevent the translation of mRNAs. In rotavirus-infected cells, even though eIF2alpha is phosphorylated these granules are not formed, suggesting that the virus prevents the assembly of these structures to allow the translation of its mRNAs. Under these conditions, some of the cellular proteins that form part of these structures were found to change their intracellular localization, with some of them having dramatic changes, like the poly(A) binding protein, which relocates from the cytoplasm to the nucleus in infected cells, a relocation that depends on the viral protein NSP3.

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (DeltaE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of DeltaE3L virus was increased by nearly 2 log(10) in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the DeltaE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in DeltaE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the alpha subunit of protein synthesis initiation factor 2 (eIF-2alpha) was elevated severalfold in DeltaE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2alpha phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with DeltaE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.

Cells have evolved elaborate mechanisms to counteract the onslaught of viral infections. To activate these defenses, the viral threat must be recognized. Danger signals, or pathogen-associated molecular patterns, that are induced by pathogens include double-stranded RNA (dsRNA), viral single-stranded RNA, glycolipids, and CpG DNA. Understanding the signal transduction pathways activated and host gene expression induced by these danger signals is vital to understanding virus-host interactions. The vaccinia virus E3L protein is involved in blocking the host antiviral response and increasing pathogenesis, functions that map to separate C-terminal dsRNA- and N-terminal Z-DNA-binding domains. Viruses containing mutations in these domains allow modeling of the role of dsRNA and Z-form nucleic acid in the host response to virus infection. Deletions in the Z-DNA- or dsRNA-binding domains led to activation of signal transduction cascades and up-regulation of host gene expression, with many genes involved in the inflammatory response. These data suggest that poxviruses actively inhibit cellular recognition of viral danger signals and the subsequent cellular response to the viral threat.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.

The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32- to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.

The most intensively studied rotavirus strains initially attach to cells when the "heads" of their protruding spikes bind cell surface sialic acid. Rotavirus strains that cause disease in humans do not bind this ligand. The structure of the sialic acid binding head (the VP8* core) from the simian rotavirus strain RRV has been reported, and neutralization epitopes have been mapped onto its surface. We report here a 1.6-A resolution crystal structure of the equivalent domain from the sialic acid-independent rotavirus strain DS-1, which causes gastroenteritis in humans. Although the RRV and DS-1 VP8* cores differ functionally, they share the same galectin-like fold. Differences between the RRV and DS-1 VP8* cores in the region that corresponds to the RRV sialic acid binding site make it unlikely that DS-1 VP8* binds an alternative carbohydrate ligand in this location. In the crystals, a surface cleft on each DS-1 VP8* core binds N-terminal residues from a neighboring molecule. This cleft may function as a ligand binding site during rotavirus replication. We also report an escape mutant analysis, which allows the mapping of heterotypic neutralizing epitopes recognized by human monoclonal antibodies onto the surface of the VP8* core. The distribution of escape mutations on the DS-1 VP8* core indicates that neutralizing antibodies that recognize VP8* of human rotavirus strains may bind a conformation of the spike that differs from those observed to date.

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.

Herpes simplex virus type 1 (HSV-1) protein ICP27 interacts with the cellular export adaptor protein Aly/REF, which is part of the exon junction complex implicated in cellular mRNA export. We previously reported that Aly/REF was no longer associated with splicing factor SC35 sites during infection but instead colocalized with ICP27 in distinct structures. Here we show that these structures colocalize with ICP4 and are sites of HSV-1 transcription. ICP27 mutants with lesions in the region required for the interaction with Aly/REF failed to recruit Aly/REF to viral transcription sites; however, ICP27 export to the cytoplasm was unimpaired, indicating that the interaction of ICP27 with Aly/REF is not required for ICP27 shuttling. ICP27 has also been shown to interact with the cellular mRNA export receptor TAP/NXF1. We report that ICP27 interacts directly with TAP/NXF1 and does not require Aly/REF to bridge the interaction. The C terminus of ICP27 is required; however, the N-terminal leucine-rich region also contributes to the interaction of ICP27 with TAP/NXF1. In contrast to the results found for Aly/REF, mutants that failed to interact with TAP/NXF1 were not exported to the cytoplasm, and TAP/NXF1 was not recruited to sites of HSV-1 transcription. Therefore, the interaction of ICP27 with TAP/NXF1 occurs after ICP27 leaves viral transcription sites. We conclude that ICP27 and the viral RNAs to which it binds are exported via the TAP/NXF1 export receptor.

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-DeltaE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2'-5'-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-DeltaE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-DeltaE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-DeltaE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein beta, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant., (Copyright 2004 American Society for Microbiology)

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

Multiple herpes simplex virus type 1 functions control translation by regulating phosphorylation of the initiation factor eIF2 on its alpha subunit. Both of the two known regulators, the gamma(1)34.5 and Us11 gene products, are produced late in the viral life cycle, although the gamma(1)34.5 gene is expressed prior to the gamma(2) Us11 gene, as gamma(2) genes require viral DNA replication for their expression while gamma(1) genes do not. The gamma(1)34.5 protein, through a GADD34-related domain, binds a cellular phosphatase (PP1alpha), maintaining pools of active, unphosphorylated eIF2. Infection of a variety of cultured cells with a gamma(1)34.5 mutant virus results in the accumulation of phosphorylated eIF2alpha and the inhibition of translation prior to the completion of the viral lytic program. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. To determine if Us11 regulates late translation, we generated two Us11 null viruses. In cells infected with a Us11 mutant, elevated levels of activated PKR and phosphorylated eIF2alpha were detected, viral translation rates were reduced 6- to 7-fold, and viral replication was reduced 13-fold compared to replication in cells infected with either wild-type virus or a virus in which the Us11 mutation was repaired. This establishes that the Us11 protein is critical for proper late translation rates. Moreover, it demonstrates that the shutoff of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus, previously ascribed solely to the gamma(1)34.5 mutation, actually results from the combined loss of gamma(1)34.5 and Us11 functions, as the gamma(2) Us11 mRNA is not translated in cells infected with a gamma(1)34.5 mutant.

Expression of the human T-cell lymphotropic virus type 1 (HTLV-1) genes is transcriptionally activated by the cognate oncoprotein Tax which enhances the binding of the cyclin AMP-responsive element binding protein (CREB) to the Tax responsive element (TxRE) located in its long terminal repeat (LTR). TxRE is highly homologous to the cyclic AMP-responsive element (CRE) except for the GC-rich sequence flanking the CRE. We cloned the cDNA for a cellular factor, TAXREB803, of which the DNA-binding domain bound to TxRE and the binding was dependent on the 3' GC-rich sequence in TxRE. TAXREB803 is an SR-related protein composed of 2,752 amino acids including numerous arginine/serine (RS) motifs. TAXREB803 enhanced both the Tax dependent transcription and the CREB binding to TxRE in cooperation with Tax. The interaction of TAXREB803 and Tax was detected by coimmunoprecipitation assays as well as by indirect immunofluorescence assays. Significantly, Tax transactivation for the HTLV-1 LTR decreased dramatically when the expression level of the endogenous TAXREB803 was suppressed by the small interfering RNA. These results suggest that TAXREB803 functions as a transcriptional coactivator for Tax and plays a critical role in the expression of HTLV-1 genes.

The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridae family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins lambda1, lambda2, and sigma2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, micro 1 and sigma3, to produce virion-like particles. Coexpression of core shell protein lambda1 and core nodule protein sigma2 was sufficient to yield CLPs that could withstand purification, whereas expression of lambda1 alone was not, indicating a required role for sigma2 as a previous study also suggested. In addition, CLPs that lacked lambda2 (formed from lambda1 and sigma2 only) could not be coated with micro 1 and sigma3, indicating a required role for lambda2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of lambda1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of lambda1 deletion mutants, we showed that the amino-terminal 230 residues of lambda1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of lambda1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of lambda1 with the threefold and/or fivefold copies of sigma2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.

Human immunodeficiency virus type 1 (HIV-1) replication requires efficient nuclear export of incompletely spliced and unspliced HIV-1 mRNA transcripts, which is achieved by Rev expression at an early stage of the viral life cycle. We have recently shown that expression of Sam68, the 68-kDa Src-associated protein in mitosis, is able to alleviate Rev function block in astrocytes by promoting Rev nuclear export. In the present study, we utilized an antisense RNA expression strategy to down-modulate constitutive Sam68 expression and examined its effect on Rev function, HIV-1 gene expression, and viral replication. These results showed that down-modulation of constitutive Sam68 expression markedly inhibited HIV-1 production in 293T cells and viral replication in T lymphocytes such as Jurkat and CEM cells, as well as human peripheral blood mononuclear cells (PBMCs). Rev-dependent in trans complementation and reporter gene assays further demonstrated that inhibition of HIV-1 gene expression by Sam68 down-modulation was due to impeded Rev activity. Moreover, digital fluorescence microscopic imaging revealed that down-modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1. Taken together, these data suggest that adequate Sam68 expression is required for Rev function and, thereby, for HIV-1 gene expression and viral replication, and they support the notion that Sam68 is directly involved in the CRM1-mediated Rev nuclear export pathway.

The function of the 5' region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5' region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5' region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

Akv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr (upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-ras by Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unr gene. All proviruses were integrated in the same transcriptional orientation as unr and N-ras genes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-ras gave rise to two different spliced products, whereas the provirus integrated into unr used only an exon skipping pattern. The absence of mutations of the N-ras codons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unr is unaffected by N-ras overexpression even in the case where the unr gene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-ras plays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unr gene unaltered.

HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/beta2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses.

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.

Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.