RNA Virus Particles Research Articles

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies.

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.

Single-particle studies of the effects of RNA–protein interactions on the self-assembly of RNA virus particles

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the invitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

Vectorial Release of Human RNA Viruses from Epithelial Cells.

Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.

Энтеровирусный везикулярный стоматит как проявление энтеровирусной инфекции. Лекция

Lesions of the oral mucosa may be a sign of systemic human diseases. Most of manifested in the oral cavity diseases are benign, but it is necessary to differentiate them from more serious diseases. Dentists should know the manifestations and recent advances in the diagnosis and treatment of oral diseases, among which are viral infections, especially highly contagious, which include hand-foot-and-mouth disease. In order to prevent its spread and to identify in time possible complications, dentist should have deep knowledge about it. Hand-foot-and-mouth disease is a highly contagious viral infectious disease caused by various enteroviruses. Viruses of the genus Enterovirus are RNA genomic. It is necessary to know the main stages of the reproduction of RNA genomic viruses in the human body. 1. Adsorption on a cell (adhesion) – an interaction between specific receptors of virions and surface molecules of sensitive cells. 2. Penetration into the cell and "undressing" can be carried out in two ways: endocytosis and translocation. 3. The synthesis of viral particles of RNA-viruses (translation) – as a result specific proteins for viral replication are synthesized. 4. Complete assembly of RNA-viruses. 5. Release of daughter virions from the cell. The source of infection can be both a sick person and a virus carrier. Asymptomatic carriage can last for weeks, sometimes months. The most intense pathogen is released in the first days of the disease. The predominant morbidity of children speaks about the high natural susceptibility of a human. For diseases, which are caused by enteroviruses, polymorphism of clinical manifestations is specific. In most cases, the violation of the general condition is insignificant and the course of the disease is benign. The initial phase is usually followed by enanthema and erythematous, papular or vesicular lesions of the skin, localized mainly on the palms and soles, as well as vesicles in the oral cavity. Enanthema usually precedes the appearance of a skin rash. Oral lesions may occur without skin symptoms. Diagnosis of enteroviral vesicular stomatitis is based on anamnestic and clinical data. There are many diagnostic tests available, but PCR of throat and vesicular fluid swabs, if available, is one of the most effective. After an illness, in most cases, immunity is formed, the possibility of developing cross-immunity to other types of the virus is not excluded

Evaluation of U.S. Food and Drug Administration Enteric Viruses Microarray for Detection of Hepatitis A Virus and Norovirus in Inoculated Tomatoes, Green Onions, and Celery

Foodborne viral contamination of fresh produce has been associated with numerous outbreaks. Detection of such contaminated foods is important in protecting public health. Here, we demonstrate for the first time the capability of the U.S. Food and Drug Administration Enteric Viruses tiling microarray (FDA-EVIR) to perform rapid molecular identification of hepatitis A virus (HAV) and human norovirus extracted from artificially inoculated fresh produce. Two published viral extraction strategies, total RNA extraction or virus particle isolation, were used to prepare the viral targets. The total RNA extraction method was used on material eluted from tomatoes, using an alkaline Tris-glycine-beef extract (TGBE) buffer. Optimization procedures including DNase treatment and poly(A)-RNA enrichment were adopted to improve microarray sensitivity. For green onions or celery, material was eluted using either glycine buffer or TGBE buffer supplemented with pectinase, respectively, and then virus particles were concentrated by ultracentrifugation. We also assessed the amount of viral RNA extracted from celery using three commercially available kits and how well that RNA performed on FDA-EVIR. Our results confirm that FDA-EVIR can identify common enteric viruses isolated from fresh produce when present as either a single or mixed species of viruses. Using total RNA extraction from tomatoes yielded a limit of detection of 1.0 × 105 genome equivalents (ge) of HAV per array input. The limit of detection for viral RNA obtained using ultracentrifugation was 1.2 × 105 ge of HAV from green onions and 1.0 × 103 ge of norovirus from celery per array input. Extending microarray methods to other food matrices should provide important support to surveillance and outbreak investigations.

Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles.

The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material.

Biochemical characterization of SARS-CoV-2 nucleocapsid protein

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients’ sera, proving the importance of this antigen in host immunity and diagnostics.

Defective Interfering Particles of Negative-Strand RNA Viruses.

Viral defective interfering particles (DIPs) were intensely studied several decades ago but research waned leaving open many critical questions. New technologies and other advances led to a resurgence in DIP studies for negative-strand RNA viruses. While DIPs have long been recognized, their exact contribution to the outcome of acute or persistent viral infections has remained elusive. Recent studies have identified defective viral genomes (DVGs) in human infections, including respiratory syncytial virus and influenza, and growing evidence indicates that DVGs influence disease severity and may contribute to viral persistence. Further, several studies have advanced our understanding of key viral and host factors that regulate DIP formation and activity. Here we review these discoveries and highlight key questions moving forward.

Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Mapping RNA-capsid interactions and RNA secondary structure within virus particles using next-generation sequencing.

To characterize RNA–capsid binding sites genome-wide within mature RNA virus particles, we have developed a Next-Generation Sequencing (NGS) platform: viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL). In vPAR-CL, 4-thiouridine is incorporated into the encapsidated genomes of virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that vPAR-CL can readily and reliably identify capsid binding sites in genomic viral RNA by detecting crosslink-specific uridine to cytidine transitions in NGS data. Using Flock House virus (FHV) as a model system, we identified highly consistent and significant vPAR-CL signals across virus RNA genome, indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites coincide with previously identified functional RNA motifs. We additionally performed dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate a high-resolution profile of single-stranded genomic RNA inside viral particles. Combining vPAR-CL and DMS-MaPseq reveals that the predominant RNA–capsid interaction sites favored double-stranded RNA regions. We disrupted secondary structures associated with vPAR-CL sites using synonymous mutations, resulting in varied effects to virus replication, propagation and packaging. Certain mutations showed substantial deficiency in virus replication, suggesting these RNA–capsid sites are multifunctional. These provide further evidence to support that FHV packaging and replication are highly coordinated and inter-dependent events.

Fecal virome composition of migratory wild duck species.

The fecal virome comprises a complex diversity of eukaryotic viruses, phages and viruses that infect the host. However, little is known about the intestinal community of viruses that is present in wild waterfowl, and the structure of this community in wild ducks has not yet been studied. The fecal virome compositions of six species of wild dabbling ducks and one species of wild diving duck were thus analyzed. Fecal samples were collected directly from the rectums of 60 ducks donated by hunters. DNA and RNA virus particles were purified and sequenced using the MiSeq Illumina platform. The reads obtained from the sequencing were analyzed and compared with sequences in the GenBank database. Viral-related sequences from the Herpesviridae, Alloherpesviridae, Adenoviridae, Retroviridae and Myoviridae viral families showed the highest overall abundances in the samples. The virome analysis identified viruses that had not been found in wild duck feces and revealed distinct virome profiles between different species and between samples of the same species. This study increases our understanding of viruses in wild ducks as possible viral reservoirs and provides a basis for further studying and monitoring the transmission of viruses from wild animals to humans and disease outbreaks in domestic animals.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Yeast killer elements hold their hosts hostage.

Yeast killer elements hold their hosts hostage.

Nucleocapsid protein from fig mosaic virus forms cytoplasmic agglomerates that are hauled by endoplasmic reticulum streaming.

Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.

Functional interplay among the flavivirus NS3 protease, helicase, and cofactors

Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses (NS2BNS3) form an endoplasmic reticulum (ER) membrane-associated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

BackgroundThe pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. Only little is known about factors influencing its general composition. Here, the effect of the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not.ResultsFrom 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads. In average, 14% of the reads showed significant sequence identities to known viruses. The percentage of detected mammalian virus sequences was highest (55–77%) in the samples of the youngest piglets and lowest (8–10%) in the samples of the sows. In contrast, the percentage of bacteriophage sequences increased from 22–44% in the youngest piglets to approximately 90% in the sows. The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and “circovirus-like” viruses CB-A and RW-A). In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group.ConclusionThe analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. Changes caused by feeding with the probiotic bacterium E. faecium could not be demonstrated using the applied metagenomics method.

A One-Step RT-PCR Array for Detection and Differentiation of Zoonotic Influenza Viruses H5N1, H9N2, and H1N1

Rapid and comprehensive pathogen identification is crucial in zoonotic influenza diagnosis. By optimizing the design of primers and probes and reverse-transcriptase polymerase chain reaction (RT-PCR) conditions, we achieved simultaneous detection of multiple influenza and zoonotic influenza viruses, including H1N1, H5N1, and H9N2 strains, in a one-step, quantitative real-time RT-PCR array (rRT-PCR array) of RNA from multiple influenza strains utilizing a single set of conditions for RT-PCR amplification. The target sequences from all targeted zoonotic influenza viruses were cloned into recombinant RNA virus particles, which were used to evaluate sensitivity, specificity, and reproducibility of the zoonotic influenza viruses RT-PCR array. The detection limit of the array was shown to be between 10(0) and 10(1) copies per reaction, and the standard curve demonstrated a linear range from 10 to 10(6) copies. Thus, the analytical sensitivity of this zoonotic influenza viruses RT-PCR array is 10-100 times higher than conventional RT-PCR. Specificity of the one-step zoonotic influenza viruses RT-PCR array was verified by comparison of results obtained with retroviral-like particles (RVPs), which contained RNA from isolates of seasonal influenza viruses, zoonotic influenza viruses, and other pathogens known to cause acute respiratory disease. The high sensitivity, rapidity, reproducibility, and specificity of this zoonotic influenza viruses rRT-PCR array has been verified as being sufficient to detect the presence of multiple zoonotic influenza viruses in a single assay. The zoonotic influenza viruses RT-PCR array might provide rapid identification of emergent zoonotic influenza viruses strains during influenza outbreaks and disease surveillance initiatives.

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.