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.     

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.     

Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.     

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.     

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.     

Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation

Cellular micro(mi)RNAs are able to recognize viral RNAs through imperfect micro-homologies. Similar to the miRNA-mediated repression of cellular translation, this recognition is thought to tether the RNAi machinery, in particular Argonaute 2 (AGO2) on viral messengers and eventually to modulate virus replication. Here, we unveil another pathway by which AGO2 can interact with retroviral mRNAs. We show that AGO2 interacts with the retroviral Group Specific Antigen (GAG) core proteins and preferentially binds unspliced RNAs through the RNA packaging sequences without affecting RNA stability or eliciting translation repression. Using RNAi experiments, we provide evidences that these interactions, observed with both the human immunodeficiency virus 1 (HIV-1) and the primate foamy virus 1 (PFV-1), are required for retroviral replication. Taken together, our results place AGO2 at the core of the retroviral life cycle and reveal original AGO2 functions that are not related to miRNAs and translation repression.     

RNA secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication

Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.     

Viral genome segmentation can result from a trade-off between genetic content and particle stability

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length.     

RNA Regulatory Elements in the Genomes of Simple Retroviruses

The RNA genomes of simple retroviruses encode three genes (gag, pol,andenv) which are required for replication. In addition, there are at least three well-definedcis-acting structures which regulate important aspects of the viral life cycle. The packaging signal at the 5′ end of the RNA tags the genomic RNA for specific encapsidation into assembling virus. Since viral Env proteins are translated from spliced mRNAs,cis-acting splicing signals ensure that the proper ratio of spliced and unspliced viral RNAs is present in the infected cell. Finally,cis-acting elements at the 3′ end of the genome promote the export of unspliced RNAs from the nucleus for translation and encapsidation.     

Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation

Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.     

Cellular protein modification by poliovirus: the two faces of poly(rC)-binding protein

During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-DeltaKH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.     

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.     

Probing the structure and function of viral RNA genomes

The majority of human, animal and plant viral pathogens possess genomes composed of RNA. The strategies evolved for expression and replication of viral RNA genomes can differ significantly from those utilized for expression and replication of host-cell genetic material. Consequently, knowledge of the molecular details of these strategies can lead to a clearer understanding of the origin, evolution and control of viral pathogens. We describe recent progress in identifying important structural and functional domains of the RNA genomes and associated replicative enzymes for two very different viruses: vesicular stomatitis virus, which possesses a single-stranded RNA genome of negative polarity, and wound tumor virus, which contains a genome composed of 12 discrete segments of double-stranded RNA.     

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.     

Tamplicon-7, a Novel T-Lymphotropic Vector Derived from Human Herpesvirus 7

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.