Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.     

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.     

Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.     

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.     

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

By dissecting and reconstituting a cell-free influenza virus genome replication system, we have purified and identified the minichromosome maintenance (MCM) complex, which is thought to be a DNA replicative helicase, as one of the host factors that regulate the virus genome replication. MCM interacted with the PA subunit of the viral RNA-dependent RNA polymerase that is found to be involved in the replication genetically. The virus genome replication was decreased in MCM2 knockdown cells. The viral polymerase appeared to be a nonproductive complex, that is, it was capable of initiating replication but produced only abortive short RNA chains. MCM stimulated de novo-initiated replication reaction by stabilizing a replication complex during its transition from initiation to elongation. Based on the findings, including the result that the MCM-mediated RNA replication reaction was competed with exogenously added RNA, we propose that MCM functions as a scaffold between the nascent RNA chains and the viral polymerase.     

Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.     

Cell Tropism Predicts Long-term Nucleotide Substitution Rates of Mammalian RNA Viruses

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001) as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively). These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.     

Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.     

Genetic stability of human T lymphotropic virus type I despite antiviral pressures by CTLs

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease. Patients with HAM/TSP show high proviral load despite increased HTLV-I Tax-specific CTL. It is still unknown whether the CTL efficiently eliminate the virus in vivo and/or whether a naturally occurring variant virus becomes predominant by escaping from the CTL. To address these issues, we sequenced a large number of HTLV-I tax genes from HLA-A*02 HAM/TSP patients and estimated synonymous and nonsynonymous changes of the genes to detect positive selection pressure on the virus. We found the pressures in three of six CTL epitopes in HTLV-I Tax, where amino acid substitutions preferentially occurred. Although some of variant viruses were not recognized by the CTL, no variant viruses accumulated within 3-8 years, indicating genetic stability of HTLV-I tax gene. These results suggest that CTL eliminate the infected cells in vivo and naturally occurring variant viruses do not predominate. As Tax is a regulatory protein which controls viral replication, the amino acid substitutions in Tax may reduce viral fitness for replication. Viral fitness and host immune response may contribute to the viral evolution within the infected individuals. Furthermore, the genetic stability in the epitopes despite the antiviral pressures suggests that the three epitopes can be the candidate targets for HTLV-I vaccine development.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.     

Estimating relative fitness in viral competition experiments

The relative fitness of viral variants has previously been defined as the slope of the logarithmic ratio of the genotype or phenotype frequencies in time plots of pairwise competition experiments. Developing mathematical models for such experiments by employing the conventional coefficient of selection s, we demonstrate that this logarithmic ratio gives the fitness difference, rather than the relative fitness. This fitness difference remains proportional to the actual replication rate realized in the particular experimental setup and hence cannot be extrapolated to other situations. Conversely, the conventional relative fitness (1 + s) should be more generic. We develop an approach to compute the generic relative fitness in conventional competition experiments. This involves an estimation of the total viral replication during the experiment and requires an estimate of the average lifetime of productively infected cells. The novel approach is illustrated by estimating the relative fitness, i.e., the relative replication rate, of a set of zidovudine-resistant human immunodeficiency virus type 1 variants. A tool for calculating the relative fitness from observed changes in viral load and genotype (or phenotype) frequencies is publically available on the website at http://www-binf.bio.uu.nl/( approximately )rdb/fitness.html.     

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.