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ABSTRACT Viruses display large variability across all stages of their life cycle, including entry, gene expression, replication, assembly, and egress. We previously reported that the immediate early adenovirus (AdV) E1A transcripts accumulate in human lung epithelial A549 cancer cells with high variability, mostly independent of the number of incoming viral genomes, but somewhat correlated to the cell cycle state at the time of inoculation. Here, we leveraged the classical Luria–Delbrück fluctuation analysis to address whether infection variability primarily arises from the cell state or stochastic noise. The E1A expression was measured by the expression of green fluorescent protein (GFP) from the endogenous E1A promoter in AdV-C5_E1A-FS2A-GFP and found to be highly correlated with the viral plaque formation, indicating reliability of the reporter virus. As an ensemble, randomly picked clonal A549 cell isolates displayed significantly higher coefficients of variation in the E1A expression than technical noise, indicating a phenotypic variability larger than noise. The underlying cell state determining infection variability was maintained for at least 9 weeks of cell cultivation. Our results indicate that preexisting cell states tune adenovirus infection in favor of the cell or the virus. These findings have implications for antiviral strategies and gene therapy applications.IMPORTANCEViral infections are known for their variability. Underlying mechanisms are still incompletely understood but have been associated with particular cell states, for example, the eukaryotic cell division cycle in DNA virus infections. A cell state is the collective of biochemical, morphological, and contextual features owing to particular conditions or at random. It affects how intrinsic or extrinsic cues trigger a response, such as cell division or anti-viral state. Here, we provide evidence that cell states with a built-in memory confer high or low susceptibility of clonal human epithelial cells to adenovirus infection. Results are reminiscent of the Luria–Delbrück fluctuation test with bacteriophage infections back in 1943, which demonstrated that mutations, in the absence of selective pressure prior to infection, cause infection resistance rather than being a consequence of infection. Our findings of dynamic cell states conferring adenovirus infection susceptibility uncover new challenges for the prediction and treatment of viral infections.

More than 100 different herpes simplex virus 1 (HSV‐1) genes belong to three major classes, and their expression is coordinately regulated and sequentially ordered in a cascade. This complex HSV‐1 gene expression is thought to be regulated by various viral and host cellular proteins. A host cellular protein, Myb‐binding protein 1A (MYBBP1A), has been reported to be associated with HSV‐1 viral genomes in conjunction with viral and cellular proteins critical for DNA replication, repair, and transcription within infected cells. However, the role(s) of MYBBP1A in HSV‐1 infections remains unclear. In this study, we examined the effects of MYBBP1A depletion on HSV‐1 infection and found that MYBBP1A depletion significantly reduced HSV‐1 replication, as well as the accumulation of several viral proteins. These results suggest that MYBBP1A is an important host cellular factor that contributes to HSV‐1 replication, plausibly by promoting viral gene expression. [ABSTRACT FROM AUTHOR]

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors. [ABSTRACT FROM AUTHOR]

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.

Epstein–Barr virus (EBV)‐associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV‐positive GC cell lines (AGS‐EBV and HGC27‐EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV‐positive GC cell line. Like SNU719, AGS‐EBV and HGC27‐EBV stably maintained their EBV genomes and expressed EBV‐encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV‐encoded miRNAs within the BamHI‐A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS‐EBV and HGC27‐EBV cells undergo distinct viral expression profiles. A very small fraction of AGS‐EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27‐EBV does not. AGS‐EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV‐positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC. [ABSTRACT FROM AUTHOR]

Infections by non-segmented negative-strand RNA viruses (NNSV) are widely thought to entail gradient gene expression from the well-established existence of a single promoter at the 3’ end of the viral genome and the assumption of constant transcriptional attenuation between genes. But multiple recent studies show viral mRNA levels in infections by respiratory syncytial virus (RSV), a major human pathogen and member of NNSV, that are inconsistent with a simple gradient. Here we integrate known and newly predicted phenomena into a biophysically reasonable model of NNSV transcription. Our model succeeds in capturing published observations of respiratory syncytial virus and vesicular stomatitis virus (VSV) mRNA levels. We therefore propose a novel understanding of NNSV transcription based on the possibility of ejective polymerase-polymerase collisions and, in the case of RSV, biased polymerase diffusion.

Background and Objectives: Among the various factors involved in the development of gastric cancer (GC), infectious agents are one of the most important causative inducers. This study aimed to investigate the possible role of EBV gene expression on SHP1 methylation in co-infection with Helicobacter pylori in patients with GC. Materials and Methods: Formalin-fixed paraffin-embedded samples were obtained from 150 patients with gastrointestinal disorders. The presence of the H. pylori and EBV genome were examined by PCR. The expression level of viral gene transcripts and methylation status of the SHP1 cellular gene was assessed by quantitative real-time PCR and methyl-specific PCR. Results: EBV and H. pylori coinfection were reported in 5.6% of patients. The mean DNA viral load was significant in patients coinfected with cagA-positive H. pylori (P= 0.02). The expression of BZLF1 and EBER was associated with GC. Also, the expression level of BZLF1in GC tissues was significantly higher in coinfection (P = 0.01). SHP1 methylation frequency was higher in the GC group than in the control group (P = 0.04). The correlation between the methylation rate and the H. pylori infection was highly significant (P<0.0001). The strongest positive correlation was observed in GC specimens between SHP1 methylation and H. pylori cagA-positive strains (p= 0.003). Conclusion: Our results suggested that cagA might involve in the elevation of EBV lytic gene expression and SHP1 methylation, and the development of gastric cancer. Understanding the mechanism of EBV H. pylori - cagA + coinfection, as well as host epigenetic changes, can play an important role in diagnosing and preventing gastric cancer. [ABSTRACT FROM AUTHOR]

Background and Objectives: Among the various factors involved in the development of gastric cancer (GC), infectious agents are one of the most important causative inducers. This study aimed to investigate the possible role of EBV gene expression on SHP1 methylation in co-infection with Helicobacter pylori in patients with GC. Materials and Methods: Formalin-fixed paraffin-embedded samples were obtained from 150 patients with gastrointestinal disorders. The presence of the H. pylori and EBV genome were examined by PCR. The expression level of viral gene transcripts and methylation status of the SHP1 cellular gene was assessed by quantitative real-time PCR and methyl-specific PCR. Results: EBV and H. pylori coinfection were reported in 5.6% of patients. The mean DNA viral load was significant in patients coinfected with cagA-positive H. pylori (P= 0.02). The expression of BZLF1 and EBER was associated with GC. Also, the expression level of BZLF1in GC tissues was significantly higher in coinfection (P = 0.01). SHP1 methylation frequency was higher in the GC group than in the control group (P = 0.04). The correlation between the methylation rate and the H. pylori infection was highly significant (P

The effect of the antiviral peptide TAT-I24 on viral gene expression in cells infected with murine cytomegalovirus (MCMV) was investigated. The expression of immediate-early, early and late genes was highly induced upon infection with MCMV. In the presence of the peptide, the expression of all tested genes was sustainably reduced to a similar extent, independent of whether they were immediate-early, early or late genes. In contrast, the expression of host genes, such as NF-κB inhibitor alpha (Nfkbia), interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), chemokine (C-X-C motif) ligand 10 (Cxcl10), chemokine (C-C motif) ligand 7 (Ccl7) and chemokine (C-C motif) ligand 5 (Ccl5), which are induced early upon virus infection, was only transiently suppressed in peptide-treated cells. The expression of other host genes which are affected by MCMV infection and play a role in endoplasmic reticulum stress or DNA-damage repair was not inhibited by the peptide. A combination of TAT-I24 with the nucleoside analogue cidofovir showed enhancement of the antiviral effect, demonstrating that viral replication can be more efficiently inhibited with a combination of drugs acting at different stages of the viral life-cycle. [ABSTRACT FROM AUTHOR]

ABSTRACT Herpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-β, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-β response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-β in cell culture. We observed that IFN-β pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-β correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-β was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-β. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0’s ability to counteract the antiviral response established by IFN-β. IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-β (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-β. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.

Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors. IMPORTANCE Infection with the respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. Most RSV studies have involved either of two historic RSV/A strains infecting one of two cell lines, HEp-2 or A549 cells. However, RSV contains ample variation within two evolving subgroups (A and B), and HEp-2 and A549 cell lines are genetically distinct. Here, we measured viral action and host response in both HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporary strains. We discovered a subgroupdependent difference in viral gene expression and found A549 cells were more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings revealed important differences between RSV subgroups and two widely used cell lines and provided baseline data for experiments with model systems better representative of natural RSV infection [ABSTRACT FROM AUTHOR]

ABSTRACT Adenoviruses are very efficient high-capacity vaccine vectors and are common gene delivery systems. Despite their extensive use in preclinical models and clinical trials over the past decades, adenoviral vectors still require optimization. To achieve that, more thorough characterizations of adenoviral genes and gene products, as well as pathogen-host interactions, are indispensable. The adenoviral DNA binding protein (DBP) is a key regulatory protein involved in various cellular and viral processes. Here, we show that single amino acid exchange mutations in human adenovirus C5 (HAdV-C5) DBP strongly influence adenoviral replication by altering interaction with the cellular ubiquitination machinery. Specifically, phenotypic analyses of DBP mutants demonstrate that single amino acid substitutions can regulate interactions with the cellular USP7 deubiquitinase, impede viral DNA synthesis, and completely abolish viral late protein expression and progeny production. Importantly, cells infected with the DBP mutant UBM5 consistently lack DBP-positive replication centers (RCs), which are usually formed during the transition from the early to the late phase of infection. Our findings demonstrate that DBP regulates a key step at the onset of the late phase of infection and that this activity is unambiguously linked to the formation and integrity of viral RCs. These data provide the experimental basis for future work that targets DBP and its interference with the formation of viral RCs during productive infection. Consequently, this work will have immediate impact on DNA virus and adenovirus research in general and, potentially, also on safety optimization of existing and development of novel adenoviral vectors and anti-adenoviral compounds. IMPORTANCE To further understand the biology of human adenoviruses (HAdVs) and to optimize HAdVs for use in prophylactic and therapeutic therapies, a thorough understanding of key viral proteins is paramount. As one of the essential HAdV proteins, the DNA binding protein DBP plays important roles in various steps of the viral replication cycle. In this work, we aimed at deciphering the role of single amino acid exchange mutations in the HAdV-C5 DBP on interaction with the cellular deubiquitinase USP7 and regulation of viral replication. We identify interaction with USP7, viral replication center formation, and viral progeny production as potently regulated steps of the viral life cycle that are affected by these few and distinct mutations in DBP.

Routine culturing of goose haemorrhagic polyomavirus (GHPV) is cumbersome, and limited data are available about its replication and gene expression profile. In this study, goose embryo fibroblast cells were infected with GHPV for temporal measurement of the viral genome copy number and mRNA levels with quantitative PCR. Accumulation of small and large tumour antigen-encoding mRNAs was detected as early as 9 hours post-infection (hpi), while high level expression of the capsid protein encoding VP1-VP3, and ORF-X mRNAs was first detected at 24 hpi. Elevation of GHPV genome copy number was noted at 48 hpi. The results indicate that the gene expression profile of GHPV is similar to that described for mammalian polyomaviruses. RESEARCH HIGHLIGHTS GHPV was propagated in culture of primary goose embryo fibroblast cells. The transcription commenced before the onset of viral DNA replication. The transcription patterns of GHPV and mammalian polyomaviruses were comparable. [ABSTRACT FROM AUTHOR]

The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2's functions as transcription modulator, activator of viral replication, and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene, which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S, which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1- and E2- induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes, which may contribute to immune evasion and HPV persistence. [ABSTRACT FROM AUTHOR]

Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts in vitro viral replication. Insertional mutagenesis of the HCMV UL30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative UL30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight UL30-associated ORFs to infection. We find that deletion of the canonically annotated UL30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replication, as we find that it reduces the expression of neighboring non-UL30-associated ORFs. Mutation of the initiating methionine of the canonical UL30 ORF indicated that it is dispensable for high- and low-MOI infection in the highly passaged AD169 strain, although it is important for low-MOI infection in the less-passaged TB40/E strain. Comutation of eight methionines in the UL30 region results in a low-MOI viral replication defect, as does mutation of the TATA box responsible for the most abundant UL30 transcript, which is found to be necessary for the accumulation of multiple UL30-associated protein isoforms during infection. In total, our data indicate the importance of the UL30-associated ORFs during low-MOI HCMV infection and further highlight the difficulty associated with the functional interrogation of broadly disruptive mutations: e.g., large deletions or transposon insertions. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

Spring viremia of carp virus (SVCV) is a globally distributed virus that causes severe clinical symptoms and high mortality in fish belonging to the families Cyprinidae and Siluridae. To protect the host against viral infection, understanding the relatedness between viral susceptibility and antiviral mechanisms must be crucial. Thus, we evaluated the viral suppression efficacy of ribavirin by measuring the transcription levels of viral and immune genes in vitro. The results showed that following ribavirin treatment after SVCV infection (MOI 0.1), ribavirin inhibited SVCV replication in epithelioma papulosum cyprini (EPC) cells and completely inhibited viral gene (G and N) expression at concentrations above 10 μg/mL at 48 h post-infection. Ribavirin does not directly damage SVCV particles but inhibits early viral replication. In the absence of SVCV infection, the immunological dynamics triggered by ribavirin resulted in upregulated pattern recognition receptors and proinflammatory cytokine-related genes (i.e., PI3K, MYD88, IRAK1, RIG-І, MAVS, Mx1, TNF-α, and NF-κB). Furthermore, EPC cells treated with ribavirin following SVCV infection showed upregulation of PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB genes within 24 h post-SVCV infection, suggesting that ribavirin positively inhibits the SVCV infection in vitro. • Ribavirin could suppress SVCV transcription by targeting the early phase of viral replication. • Ribavirin-exposed EPC cells had less SVCV intracellularly and could maintain the cell morphology. • Ribavirin upregulated the innate immune genes, PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB in the SVCV-infected cell. [ABSTRACT FROM AUTHOR]

Abstract The lymphocystis disease (LCD), the main viral pathology described in cultured gilthead seabream (Sparus aurata), is a self-limiting condition characterized by the appearance of hypertrophied fibroblasts (named lymphocysts) in the connective tissue of fish, primarily in the skin and fins. The causative agent of the disease is the Lymphocystis disease virus (LCDV), a member of the Iridoviridae family. In the present study, LCDV genome and transcripts were detected by real-time PCR in caudal fin, as well as in several internal organs, such as intestine, liver, spleen, kidney and brain, from asymptomatic, diseased and recovered gilthead seabream juveniles. These results indicate that the LCDV has a broad range tissue tropism, and can establish a systemic infection, even in subclinically infected fish. As showed by in situ hybridization, the permissive cells for LCDV infection seem to be fibroblasts, hepatocytes and cells of the mononuclear phagocyte system. Histopathological alterations associated with LCD were observed in all the organs analysed, including necrotic changes in liver and kidney, inflammatory response in the intestine submucosa or brain haemorrhage, although lymphocysts were only detected in the dermis of the caudal fin. Nevertheless, these histological changes were reverted in recovered animals.

Bovine herpesvirus 5 is an alphaherpesvirus that causes nonsuppurative meningoencephalitis in cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Although previous studies elucidated crucial aspects involved in the pathogenesis of the disease, there is a paucity of information regarding the molecular events contributing to infection and replication of BoHV-5. The objective of the present study was to determine the in vitro gene expression pattern of BoHV-5 (e.g., alpha, beta, and gamma genes) and host cells genes (GAPDH and 18S) over time utilizing different quantities of inoculated virus. Three BoHV-5 genes (bICP0, UL9, US4) and one structural bovine cell gene had their expression accessed by real-time PCR. While the expression of BoHV-5 genes increased during the course of infection, GAPDH gene expression decreased in the host cells, evidencing the effect of viral infection on the expression of bovine cell genes. The 18S ribosomal RNA (rRNA) gene was constitutively expressed throughout BoHV-5 infection. Our data clearly demonstrates that GAPDH gene should not be used as a reference gene in studies of BoHV-5 infection because it was influenced by viral infection. However, 18S rRNA was constitutively expressed and, therefore, is recommended for normalization of BoHV-5 infection studies in bovine cells. The expression of viral genes transcripts was not altered by increasing number of viral particles added to the culture. All viral genes included here demonstrated the same expression pattern over time and there was no difference in the expression of viral genes among the various time points. Our data show important differences comparing to classical studies regarding herpesvirus alpha, beta, and gamma genes expression. More research is necessary to improve our understanding about the BoHV-5 biology during infection. Studies employing next-generation sequencing (i.e., RNA-seq), using both in vitro and in vivo models, would be the next logical step to grasp the virus and host cell’s transcriptome changes over the course of infection.

Introduction: Mucosal immune activation, in the context of sexual transmission of HIV-1 infection, is crucial, as the increased presence of activated T cells enhance susceptibility to infection. In this regard, it has been proposed that immunomodulatory compounds capable of modulating immune activation, such as Vitamin D (VitD) may reduce HIV-1 transmission and might be used as a safe and cost-effective strategy for prevention. Considering this, we examined the in vitro effect of the treatment of peripheral blood mononuclear cells (PBMCs) with the active form of VitD, calcitriol, on cellular activation, function and susceptibility of CD4+ T cells to HIV-1 infection. Methods: We treated PBMCs from healthy HIV unexposed individuals (Co-HC) and frequently exposed, HIV-1 seronegative individuals (HESNs) from Colombia and from healthy non-exposed individuals from Canada (Ca-HC) with calcitriol and performed in vitro HIV-1 infection assays using X4- and R5-tropic HIV-1 strains respectively. In addition, we evaluated the activation and function of T cells and the expression of viral co-receptors, and select antiviral genes following calcitriol treatment. Results: Calcitriol reduced the frequency of infected CD4+ T cells and the number of viral particles per cell, for both, X4- and R5-tropic viruses tested in the Co-HC and the Ca-HC, respectively, but not in HESNs. Furthermore, in the Co-HC, calcitriol reduced the frequency of polyclonally activated T cells expressing the activation markers HLA-DR and CD38, and those HLA-DR+CD38-, whereas increased the subpopulation HLA-DR-CD38+. Calcitriol treatment also decreased production of granzyme, IL-2 and MIP-1β by T cells and increased the transcriptional expression of the inhibitor of NF-kB and the antiviral genes cathelicidin (CAMP) and APOBEC3G in PBMCs from Co-HC. Conclusion: Our in vitro findings suggest that VitD treatment could reduce HIV-1 transmission through a specific modulation of the activation levels and function of T cells, and the production of antiviral factors. In conclusion, VitD remains as an interesting potential strategy to prevent HIV-1 transmission that should be further explored. [ABSTRACT FROM AUTHOR]

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses. [ABSTRACT FROM AUTHOR]

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis. [ABSTRACT FROM AUTHOR]

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs. [ABSTRACT FROM AUTHOR]

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion. [ABSTRACT FROM AUTHOR]

Precise promoter annotation is required for understanding the mechanistic basis of transcription initiation. In the context of complex genomes, such as herpesviruses where there is extensive genic overlap, identification of transcription start sites (TSSs) is particularly problematic and cannot be comprehensively accessed by standard RNA sequencing approaches. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of Kaposi’s sarcoma and the B cell lymphoma primary effusion lymphoma (PEL). Here, we leverage RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) and define KSHV TSSs transcriptome-wide and at nucleotide resolution in two widely used models of KSHV infection, namely iSLK.219 cells and the PEL cell line TREx-BCBL1-RTA. By mapping TSSs over a 96 h time course of reactivation we confirm 48 of 50 previously identified TSSs. Moreover, we identify over 100 novel transcription start site clusters (TSCs) in each cell line. Our analyses identified cell-type specific differences in TSC positions as well as promoter strength, and defined motifs within viral core promoters. Collectively, by defining TSSs at high resolution we have greatly expanded the transcriptional landscape of the KSHV genome and identified transcriptional control mechanisms at play during KSHV lytic reactivation. [ABSTRACT FROM AUTHOR]

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi’s sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level. [ABSTRACT FROM AUTHOR]

The article discusses the formation of herpes simplex virus (HSV) replication compartments (RC), as well as their ability to attract cellular and viral proteins and the effect of the interactions between distinct individual RCs. Topics include viral genome amplification, the complex virus-host interactions, and the three stages of HSV-1 viral gene expression, namely, immediate early (IE), early (E), and late (L).

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development. [ABSTRACT FROM AUTHOR]

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies. [ABSTRACT FROM AUTHOR]

Perinatal viral infections can cause neurodevelopmental disorders with varying degrees of cognitive and mental sequelae. One virus that affects neurodevelopment is the Zika Virus (ZIKV), which can cause microcephaly and other congenital malformations during pregnancy. Despite the severity of neurodevelopmental impairments associated with ZIKV infection during pregnancy, little is known about the neuropathogenic mechanisms responsible for central nervous system malformations and dysfunctions. In this study, our main objective was to investigate a ZIKV infection model using human neural progenitor cells, specifically the hNS-1 cell line, which can differentiate into neurons, astrocytes, and oligodendrocytes. We also evaluated neural differentiation by measuring the expression of GFAP (astrocyte marker) and β-Tubulin III (neuron marker) in undifferentiated hNS-1 cells infected with ZIKV. We found increased GFAP messenger RNA expression in infected cells at 9 and 15 days of differentiation, suggesting changes in the astrocyte population. Based on these changes in GFAP expression, we isolated the astrocyte population from a 21-day differentiated hNS-1 cell culture and demonstrated that ZIKV infection reduces cell viability, increases the production of reactive oxygen species (ROS), and results in high viral titers. Additionally, we observed changes in the expression of genes involved in viral entry into cells and genes related to glutamatergic system homeostasis. Our findings provide new evidence on how ZIKV infects neural progenitor cells and its dependence on cell differentiation. We also highlight the modification of GFAP expression and the potential functionality of astrocytes. These results contribute to a better understanding of the pathophysiology of congenital ZIKV-associated disease. [ABSTRACT FROM AUTHOR]

MDA5 belongs to the RIG-I-like receptor family and plays a non-redundant role in recognizing cytoplasmic viral RNA to induce the production of type I IFNs. Upon RNA ligand stimulation, we observed the redistribution of MDA5 from the cytosol to mitochondrial membrane fractions. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is an essential accessory protein for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may interact with MDA5 through the CARDs (N-MDA5), but 14-3-3η was the only isoform that could enhance MDA5-dependent IFNβ promoter activities in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells impaired and delayed the kinetics of MDA5 oligomerization, which is a critical step for MDA5 activation. Consequently, the MDA5-dependent IFNβ promoter activities as well as IFNβ mRNA expression level were also decreased in the 14-3-3η knocked-down cells. We also demonstrated that 14-3-3η is essential in boosting the activation of MDA5-dependent antiviral innate immunity during viral infections. In conclusion, our results uncover a novel function of 14-3-3η to promote the MDA5-dependent IFNβ induction pathway by reducing the immunostimulatory potential of viral dsRNA within MDA5 activation signaling pathway. [ABSTRACT FROM AUTHOR]

Cotton is a commercial and economically important crop that generates billions of dollars in annual revenue worldwide. However, cotton yield is affected by a sap-sucking insect Bemisia tabaci (whitefly), and whitefly-borne cotton leaf curl disease (CLCuD). The causative agent of devastating CLCuD is led by the viruses belonging to the genus Begomovirus (family Geminiviridae), collectively called cotton leaf curl viruses. Unfortunately, the extensively cultivated cotton (Gossypium hirsutum) species are highly susceptible and vulnerable to CLCuD. Yet, the concomitant influence of whitefly and CLCuD on the susceptible G. hirsutum transcriptome has not been interpreted. In the present study we have employed an RNA Sequencing (RNA-Seq) transcriptomics approach to explore the differential gene expression in susceptible G. hirsutum variety upon infection with viruliferous whiteflies. Comparative RNA-Seq of control and CLCuD infected plants was done using Illumina HiSeq 2500. This study yielded 468 differentially expressed genes (DEGs). Among them, we identified 220 up and 248 downregulated DEGs involved in disease responses and pathogen defense. We selected ten genes for downstream RT-qPCR analyses on two cultivars, Karishma and MNH 786 that are susceptible to CLCuD. We observed a similar expression pattern of these genes in both susceptible cultivars that was also consistent with our transcriptome data further implying a wider application of our global transcription study on host susceptibility to CLCuD. We next performed weighted gene co-expression network analysis that revealed six modules. This analysis also identified highly co-expressed genes as well as 55 hub genes that co-express with ≥ 50 genes. Intriguingly, most of these hub genes are shown to be downregulated and enriched in cellular processes. Under-expression of such highly co-expressed genes suggests their roles in favoring the virus and enhancing plant susceptibility to CLCuD. We also discuss the potential mechanisms governing the establishment of disease susceptibility. Overall, our study provides a comprehensive differential gene expression analysis of G. hirsutum under whitefly-mediated CLCuD infection. This vital study will advance the understanding of simultaneous effect of whitefly and virus on their host and aid in identifying important G. hirsutum genes which intricate in its susceptibility to CLCuD. [ABSTRACT FROM AUTHOR]

Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR. [ABSTRACT FROM AUTHOR]

Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed “host shutoff.” Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We analyzed viral growth, general shutoff activity, specificity of mRNA targets, and viral gene expressions. Our results showed that PA-X was the major contributor in reducing general host protein expression in the virus-infected cells. Intriguingly, our transcriptomic analysis from infected human airway A549 cells indicate that shutoff-active NS1 specifically targeted host mRNAs related to interferon (IFN) signaling pathways and cytokine release. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of shutoff-active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing shutoff-active NS1 with reduced amount of PA-X expression most efficiently suppressed antiviral and innate immune responses in human cells, indicating that influenza virus needs to optimize the contribution of these two shutoff proteins to circumvent host responses for its optimum growth. [ABSTRACT FROM AUTHOR]

Orthobunyaviruses such as Cache Valley virus (CVV) and Kairi virus (KRIV) are important animal pathogens. Periodic outbreaks of CVV have resulted in the significant loss of lambs on North American farms, whilst KRIV has mainly been detected in South and Central America with little overlap in geographical range. Vaccines or treatments for these viruses are unavailable. One approach to develop novel vaccine candidates is based on the use of reverse genetics to produce attenuated viruses that elicit immune responses but cannot revert to full virulence. The full genomes of both viruses were sequenced to obtain up to date genome sequence information. Following sequencing, minigenome systems and reverse genetics systems for both CVV and KRIV were developed. Both CVV and KRIV showed a wide in vitro cell host range, with BHK-21 cells a suitable host cell line for virus propagation and titration. To develop attenuated viruses, the open reading frames of the NSs proteins were disrupted. The recombinant viruses with no NSs protein expression induced the production of type I interferon (IFN), indicating that for both viruses NSs functions as an IFN antagonist and that such attenuated viruses could form the basis for attenuated viral vaccines. To assess the potential for reassortment between CVV and KRIV, which could be relevant during vaccination campaigns in areas of overlap, we attempted to produce M segment reassortants by reverse genetics. We were unable to obtain such viruses, suggesting that it is an unlikely event. [ABSTRACT FROM AUTHOR]

Overlapping genes represent a fascinating evolutionary puzzle, since they encode two functionally unrelated proteins from the same DNA sequence. They originate by a mechanism of overprinting, in which point mutations in an existing frame allow the expression (the "birth") of a completely new protein from a second frame. In viruses, in which overlapping genes are abundant, these new proteins often play a critical role in infection, yet they are frequently overlooked during genome annotation. This results in erroneous interpretation of mutational studies and in a significant waste of resources. Therefore, overlapping genes need to be correctly detected, especially since they are now thought to be abundant also in eukaryotes. Developing better detection methods and conducting systematic evolutionary studies require a large, reliable benchmark dataset of known cases. We thus assembled a high-quality dataset of 80 viral overlapping genes whose expression is experimentally proven. Many of them were not present in databases. We found that overall, overlapping genes differ significantly from non-overlapping genes in their nucleotide and amino acid composition. In particular, the proteins they encode are enriched in high-degeneracy amino acids and depleted in low-degeneracy ones, which may alleviate the evolutionary constraints acting on overlapping genes. Principal component analysis revealed that the vast majority of overlapping genes follow a similar composition bias, despite their heterogeneity in length and function. Six proven mammalian overlapping genes also followed this bias. We propose that this apparently near-universal composition bias may either favour the birth of overlapping genes, or/and result from selection pressure acting on them. [ABSTRACT FROM AUTHOR]

G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we accessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression. [ABSTRACT FROM AUTHOR]

To elucidate HIV-1 co-infection-induced acceleration of HCV liver disease and identify stage-specific molecular signatures, we applied a new high-resolution molecular screen, the Affymetrix GeneChip Human Transcriptome Array (HTA2.0), to HCV-mono- and HIV/HCV-co-infected liver specimens from subjects with early and advanced disease. Out of 67,528 well-annotated genes, we have analyzed the functional and statistical significance of 75 and 28 genes expressed differentially between early and advanced stages of HCV mono- and HIV/HCV co-infected patient liver samples, respectively. We also evaluated the expression of 25 and 17 genes between early stages of mono- and co-infected liver tissues and between advanced stages of mono- and co-infected patient’s samples, respectively. Based on our analysis of fold-change in gene expression as a function of disease stage (i.e., early vs. advanced), coupled with consideration of the known relevant functions of these genes, we focused on four candidate genes, ACSL4, GNMT, IFI27, and miR122, which are expressed stage-specifically in HCV mono- and HIV-1/HCV co-infective liver disease and are known to play a pivotal role in regulating HCV-mediated hepatocellular carcinoma (HCC). Our qRT-PCR analysis of the four genes in patient liver specimens supported the microarray data. Protein products of each gene were detected in the endoplasmic reticulum (ER) where HCV replication takes place, and the genes' expression significantly altered replicability of HCV in the subgenomic replicon harboring regulatory genes of the JFH1 strain of HCV in Huh7.5.1. With respect to three well-known transferrable HIV-1 viral elements—Env, Nef, and Tat—Nef uniquely augmented replicon expression, while Tat, but not the others, substantially modulated expression of the candidate genes in hepatocytic cells. Combinatorial expression of these cellular and viral genes in the replicon cells further altered replicon expression. Taken together, these results showed that HIV-1 viral proteins can exacerbate liver pathology in the co-infected patients by disparate molecular mechanisms—directly or indirectly dysregulating HCV replication, even if lack of association of HCV load and end-stage liver disease in hemophilic patients were reported, and modulating expression of hepatocellular genes critical for disease progression. These findings also provide major insights into development of stage-specific hepatocellular biomarkers for improved diagnosis and prognosis of HCV-mediated liver disease. [ABSTRACT FROM AUTHOR]

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses—EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses—in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes. [ABSTRACT FROM AUTHOR]

Cotesia plutellae (= vestalis) bracovirus (CpBV) is symbiotic to an endoparasitoid wasp, C. plutellae, and plays crucial roles in parasitism against the diamondback moth, Plutella xylostella. CpBV virion genome consists of 35 circular DNAs encoding 157 putative open reading frames (ORFs). This study re-annotated 157 ORFs with update genome database and analyzed their gene expressions at early and late parasitic stages. Re-annotation has established 15 different viral gene families, to which 83 ORFs are assigned with remaining 74 hypothetical genes. Among 157 ORFs, 147 genes were expressed at early or late parasitic stages, among which 141 genes were expressed in both parasitic stages, indicating persistent nature of gene expression. Relative frequencies of different viral circles present in the ovarian lumen did not explain the expression variation of the viral ORFs. Furthermore, expression level of each viral gene was varied during parasitism along with host development. Highly up-regulated CpBV genes at early parasitic stage included BEN (ANP, 5R and AC1), ELP (EP1-like protein), IkB (inhibitor kB), P494 (protein 494 kDa) family genes, while those at late stage were mostly hypothetical genes. Along with the viral gene expression, 362 host genes exhibited more than two fold changes in expression levels at early parasitic stage compared to nonparasitized host. At late stage, more number (1,858) of host genes was regulated. These results suggest that persistent expression of most CpBV genes may be necessary to regulate host physiological processes during C. plutellae parasitism. [ABSTRACT FROM AUTHOR]

Maize Iranian mosaic virus (MIMV; Mononegavirales, Rhabdoviridae, Nucleorhabdovirus) infects maize and several other poaceous plants. MIMV encodes six proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). In the present study, MIMV gene expression and genetic polymorphism of an MIMV population in maize were determined. N, P, P3, and M protein genes were more highly expressed than the 5′ terminal G and L genes. Twelve single nucleotide polymorphisms were identified across the genome within a MIMV population in maize from RNA-Seq read data pooled from three infected plants indicating genomic variations of potential importance to evolution of the virus. MIMV N, P, and M proteins that are known to be involved in rhabdovirus replication and transcription were characterized as to their intracellular localization and interactions. N protein accumulated exclusively in the nucleus and interacted with itself and with P protein. P protein accumulated in both the nucleus and cell periphery and interacted with itself, N and M proteins in the nucleus. M protein was localized in the cell periphery and on endomembranes, and interacted with P protein in the nucleus. MIMV proteins show a distinctive combination of intracellular localizations and interactions. [ABSTRACT FROM AUTHOR]