Positive-strand RNA virus genome replication organelles: structure, assembly, control.

Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.

Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching.

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication.

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand.

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesized that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed, this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV), and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3.

The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication.

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.

A novel NS3/4A protease dependent cleavage site within pestiviral NS2.

Pestiviruses like bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae. A special feature of the Flaviviridae is the importance of nonstructural (NS) proteins for both genome replication and virion morphogenesis. The NS2-3-4A region and its regulated processing by the NS2 autoprotease and the NS3/4A protease plays a central role in the pestiviral life cycle. We report the identification and characterization of a novel internal cleavage in BVDV NS2, which is mediated by the NS3/4A protease. Further mapping using the NS2 of BVDV-1 strain NCP7 showed that cleavage occurs between L188 and G189. This cleavage site represents a novel sequence motif recognized by the NS3/4A protease and is conserved between the pestivirus species A, B and D. Inhibition of this internal NS2 cleavage by mutating the cleavage site did not cause obvious effects on RNA replication or virion morphogenesis in cultured cell lines. Accordingly, this novel internal NS2 cleavage adds an additional layer to the already complex polyprotein processing of Pestiviruses and might further extend the repertoires of the multifunctional NS2. However, unravelling of the functional relevance of this novel processing event in NS2, therefore, awaits future in vivo studies.

2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation.

For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses.

Pestiviruses like bovine viral diarrhea virus (BVDV) are a threat to livestock. For pestiviruses, cytopathogenic (cp) and noncytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections, which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2, the activity of which depends on its cellular cofactor, DNAJC14. Since this chaperone is available in small amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cp BVDV strains arising mostly by RNA recombination show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses has been established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14 dependent. By generating bovine and porcine DNAJC14 knockout cells, we could show that (i) replication of 6 distinct noncp pestivirus species (A to D, F, and G) depends on DNAJC14, (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14, and (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as a pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.IMPORTANCE Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo.

Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5' untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps.IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles.

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

Glycine Zipper Motifs in Hepatitis C Virus Nonstructural Protein 4B Are Required for the Establishment of Viral Replication Organelles.

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.

Characterization of a Threonine-Rich Cluster in Hepatitis C Virus Nonstructural Protein 5A and Its Contribution to Hyperphosphorylation.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein with key functions in regulating viral RNA replication and assembly. Two phosphoisoforms are discriminated by their different apparent molecular weights: a basally phosphorylated (p56) and a hyperphosphorylated (p58) variant. The precise mechanisms governing p58 synthesis and specific functions of the isoforms are poorly understood. Our study aimed at a deeper understanding of determinants involved in p58 synthesis. We analyzed two variants of p56 and p58 of isolate JFH-1 separately by mass spectrometry using an expression model and thereby identified a threonine-rich phosphopeptide exclusively found in the hyperphosphorylated variant. Individual exchange of possible phosphoacceptor sites to phosphoablatant or -mimetic residues had little impact on HCV replication or assembly in cell culture. A phosphospecific antibody recognizing pT242 revealed that this position was indeed phosphorylated only in p58 and depended on casein kinase Iα. Importantly, phosphoablative mutations at positions T244 and S247 abrogated pT242 detection without substantial effects on global p58 levels, whereas mutations in the preceding serine-rich cluster dramatically reduced total p58 levels but had minor impact on pT242 levels, suggesting the existence of distinct subspecies of hyperphosphorylated NS5A. Mass spectrometry analyses of different genotypes showed variable phosphorylation patterns across NS5A and suggested that the threonine-rich region is also phosphorylated at T242 in gt4a and at S249 in gt1a, gt1b, and gt4a. Our data therefore indicate that p58 is not a single homogenously phosphorylated protein species but rather a population of various phosphoisoforms, with high variability between genotypes.IMPORTANCE Hepatitis C virus infections affect 71 million people worldwide and cause severe chronic liver disease. Recently, efficient antiviral therapies have been established, with inhibitors of nonstructural protein NS5A as a cornerstone. NS5A is a central regulator of HCV replication and assembly but is still enigmatic in its molecular functions. It exists in two phosphoisoforms, p56 and p58. We identified a phosphopeptide exclusively found in p58 and analyzed the determinants involved in phosphorylation of this region. We found evidence for very different phosphorylation patterns resulting in p58. These results challenge the concept of p58 being a homogenous species of NS5A molecules phosphorylated at the same positions and argues for at least two independently phosphorylated variants showing the same electrophoretic mobility, likely serving different functions.

Update on hepatitis E virology: Implications for clinical practice.

Hepatitis E virus (HEV) is a positive-strand RNA virus transmitted by the fecal-oral route. The 7.2kb genome encodes three open reading frames (ORF) which are translated into (i) the ORF1 polyprotein, representing the viral replicase, (ii) the ORF2 protein, corresponding to the viral capsid, and (iii) the ORF3 protein, a small protein involved in particle secretion. Although HEV is a non-enveloped virus in bile and feces, it circulates in the bloodstream wrapped in cellular membranes. HEV genotypes 1 and 2 infect only humans and cause mainly waterborne outbreaks. HEV genotypes 3 and 4 are widely represented in the animal kingdom and are transmitted as a zoonosis mainly via contaminated meat. HEV infection is usually self-limited but may persist and cause chronic hepatitis in immunocompromised patients. Reduction of immunosuppressive treatment or antiviral therapy with ribavirin have proven effective in most patients with chronic hepatitis E but therapy failures have been reported. Alternative treatment options are needed, therefore. Infection with HEV may also cause a number of extrahepatic manifestations, especially neurologic complications. Progress in the understanding of the biology of HEV should contribute to improved control and treatment of HEV infection.

Viruses and the nucleolus: the fatal attraction.

Viruses are small obligatory parasites and as a consequence, they have developed sophisticated strategies to exploit the host cell's functions to create an environment that favors their own replication. A common feature of most - if not all - families of human and non-human viruses concerns their interaction with the nucleolus. The nucleolus is a multifunctional nuclear domain, which, in addition to its well-known role in ribosome biogenesis, plays several crucial other functions. Viral infection induces important nucleolar alterations. Indeed, during viral infection numerous viral components localize in nucleoli, while various host nucleolar proteins are redistributed in other cell compartments or are modified, and non-nucleolar cellular proteins reach the nucleolus. This review highlights the interactions reported between the nucleolus and some human or animal viral families able to establish a latent or productive infection, selected on the basis of their known interactions with the nucleolus and the nucleolar activities, and their links with virus replication and/or pathogenesis. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

Self-Replicating RNA Derived from the Genomes of Positive-Strand RNA Viruses.

Self-replicating RNA derived from the genomes of positive-strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.

Activation of MAPK-mediated immunity by phosphatidic acid in response to positive-strand RNA viruses.

Increasing evidence suggests that mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant defense against viruses. However, the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear. In this study, we discovered that phosphatidic acid (PA) represents a major class of lipids that respond to Potato virus Y (PVY) at an early stage of infection. We identified NbPLDα1 (Nicotiana benthamiana phospholipase Dα1) as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role. 6K2 of PVY interacts with NbPLDα1, leading to elevated PA levels. In addition, NbPLDα1 and PA are recruited by 6K2 to membrane-bound viral replication complexes. On the other hand, 6K2 also induces activation of the MAPK pathway, dependent on its interaction with NbPLDα1 and the derived PA. PA binds to WIPK/SIPK/NTF4, prompting their phosphorylation of WRKY8. Notably, spraying with exogenous PA is sufficient to activate the MAPK pathway. Knockdown of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA. 6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDα1 and induced the activation of MAPK-mediated immunity. Loss of function of NbPLDα1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation. Thus, activation of MAPK-mediated immunity by NbPLDα1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.