Search

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89’s structure may mediate its function., Author summary HCMV is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients for which the development of new non-nucleosidic antiviral agents are highly important. This manuscript focuses on DNA packaging, which is a target for development of new antivirals. The terminase subunit pUL89 is involved in this process. The paper presents the identification of DNA binding and nuclease motifs with invariant amino acids and highlights its first 3-D surface structure at approx. 3 nm resolution. At this resolution, the calculated 3-D surface structure matches well with the predicted structure. In conjunction with earlier studies it was possible to define structure-function relationships for the HCMV terminase subunit pUL89.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication., Author summary Replication of positive-strand RNA viruses, which infect plants and animals, depends on many cellular resources. These viruses subvert cellular membranes and co-opt host proteins to build replication compartments that produce the viral progeny. The viral replication process also requires cellular metabolites and energy in the form of ATP. Using plant host as well as the model host yeast, the authors discovered that tomato bushy stunt tombusvirus (TBSV) co-opts the fermentation pathway to facilitate the local generation of ATP within the replication compartments. TBSV replication induces high expression of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes. With the help of the viral replication protein, TBSV recruits Pdc1 and Adh1 into the viral replication compartment. The authors propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses by facilitating rapid and efficient replication in the infected hosts. The authors also showed that the replication of five other plant viruses depends on the fermentation pathway in plants.

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNβ pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that–in contrast to cardiomyocytes–other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection., Author summary Viruses can infect the heart, causing inflammation–termed myocarditis–which is a serious, and sometimes fatal, disease. One way to combat the infection is by stimulating our immune system, encouraging it to fight the virus. However, the treatment that is currently used “revs up” many different parts of our immune system, including some that play little or no role in clearing the virus, and this wide-ranging activation increases the risk of potentially-harmful side effects. We want to identify the parts of the immune system that fight virus infections of the heart, so that we can improve the treatment of viral myocarditis by selectively stimulating only those immune responses, thereby retaining the benefit of treatment (i.e., clearing the virus) while reducing its cost (i.e. lowering the risk of harmful side effects). In this paper, we demonstrate that a family of proteins called IFITs play a role in protecting many tissues against these infections, but are particularly important in heart muscle cells, in which they are indispensable. Thus, IFITs represent a possible target for the treatment of viral myocarditis.

Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5’RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5’RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5’RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection., Author summary RNA silencing and RNA decay are two essential pathways that determine the fate of cellular RNA molecules. RNA decay has been suggested to compete with RNA silencing for the RNA substrates and thus to suppress RNA silencing in plants. In this report, we show that the deficiency of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) rather than overexpression of them attenuates RNA silencing and facilitates viral RNA accumulation in turnip mosaic virus (TuMV)-infected Nicotiana benthamiana plants. 5’RDGs can inhibit the accumulation of exogenous gene transcripts or viral RNA via the RNA decay pathway when RNA silencing is compromised. Therefore, RNA silencing and RNA decay are important defense mechanisms that coordinate to function against exogenous gene transcripts or viral RNA. It is well known that viruses have evolved to encode viral suppressors of RNA silencing (VSRs) that counteract RNA silencing for an invading virus to establish infection. We discover that two known VSRs of TuMV interact with 5’RDGs and the interactions subvert RNA decay. Taken together, this study provides new insights into the concerted function of RNA silencing and RNA decay in plant immunity against exogenous gene transcripts and viral RNA, and reveals a novel function of VSRs.

Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment., Author summary Viral replication organelles are formed in subcellular compartments during positive-strand RNA virus infections to support robust virus replication. TBSV induces multivesicular body-like structures consisting of aggregated peroxisomes. However, endoplasmic reticulum (ER) and early endosomal proteins and membranes also contribute to the biogenesis of the replication compartment. The authors show that the syntaxin 18-like Ufe1 and Use1 ER SNARE proteins, which are present in ER subdomains called ERAS (ER arrival site), are necessary for the formation of the viral replication organelles. By binding to the p33 replication protein of TBSV, Ufe1 and Use1 serve as an assembly hub for biogenesis of the replication compartment and facilitating the transfer of phospholipids and sterols to the growing sites of viral replication. The advantage of co-opting ER resident SNAREs could be that these proteins constitute very active ER subdomains (ERAS), which might be especially suitable for generation of the extensive membranous viral replication compartment.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst., Author summary Infectious bacteria have many strategies to weaken the defenses of their hosts. One common strategy is to inject proteins called effectors into the host cells. Many effectors inactivate proteins that transmit defense signals, disabling the alarm systems that alert the plant to defend against the pathogen. In this paper, we show that pathogens can also target very important small molecules in eukaryotes. We found that the plant pathogen effector AvrRxo1 adds a modification to NAD, a molecule required for hundreds of respiratory and signaling reactions, when expressed inside yeast or plant cells. This modification produces an unusual molecule, 3’-NADP, that is not able to be used efficiently by the cell. This finding demonstrates a new way that effectors can target essential central metabolites to manipulate the host.

In eukaryotes, the degradation of cellular mRNAs is accomplished by Xrn1 and the cytoplasmic exosome. Because viral RNAs often lack canonical caps or poly-A tails, they can also be vulnerable to degradation by these host exonucleases. Yeast lack sophisticated mechanisms of innate and adaptive immunity, but do use RNA degradation as an antiviral defense mechanism. We find a highly refined, species-specific relationship between Xrn1p and the “L-A” totiviruses of different Saccharomyces yeast species. We show that the gene XRN1 has evolved rapidly under positive natural selection in Saccharomyces yeast, resulting in high levels of Xrn1p protein sequence divergence from one yeast species to the next. We also show that these sequence differences translate to differential interactions with the L-A virus, where Xrn1p from S. cerevisiae is most efficient at controlling the L-A virus that chronically infects S. cerevisiae, and Xrn1p from S. kudriavzevii is most efficient at controlling the L-A-like virus that we have discovered within S. kudriavzevii. All Xrn1p orthologs are equivalent in their interaction with another virus-like parasite, the Ty1 retrotransposon. Thus, Xrn1p appears to co-evolve with totiviruses to maintain its potent antiviral activity and limit viral propagation in Saccharomyces yeasts. We demonstrate that Xrn1p physically interacts with the Gag protein encoded by the L-A virus, suggesting a host-virus interaction that is more complicated than just Xrn1p-mediated nucleolytic digestion of viral RNAs., Author Summary Like other eukaryotes, Saccharomyces cerevisiae is chronically infected with viruses. It is fascinating to consider how S. cerevisiae deals with viral infection, because yeast have limited mechanisms of immunity. Our paper focuses on Xrn1p, an enzyme that is important for the destruction of irregular cellular RNAs in all eukaryotic cells. Xrn1p also degrades viral RNAs, owing to the fact that viral RNAs share biochemical characteristics with aberrant cellular mRNAs. Xrn1p was previously known to efficiently control the replication of a S. cerevisiae virus called “L-A.” We find that two different L-A viruses of Saccharomyces yeasts are best controlled by the Xrn1p from their own host species compared to the Xrn1p from other species. Importantly, these Xrn1p from different species are functionally equivalent in all other ways. This would suggest that while the important cellular functions of Xrn1p have been conserved over millions of years, the interaction with L-A-like viruses has been dynamic and constantly redefined by evolution. The identification of species-specific host proteins, like Xrn1p, is recently being appreciated as a key criterion for understanding why viruses infect the species that they do.