The 5′UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins

Foy

et al. 2011

Self Cite

The Alphavirus genus in the Togaviridae family contains a number of human and animal pathogens. The importance of alphaviruses has been strongly underappreciated; however, epidemics of chikungunya virus (CHIKV), causing millions of cases of severe and often persistent arthritis in the Indian subcontinent, have raised their profile in recent years. In spite of a continuous public health threat, to date no licensed vaccines have been developed for alphavirus infections. In this study, we have applied an accumulated knowledge about the mechanism of alphavirus replication and protein function in virus-host interactions to introduce a new approach in designing attenuated alphaviruses. These variants were constructed from genes derived from different, geographically isolated viruses. The resulting viable variants encoded CHIKV envelope and, in contrast to naturally circulating viruses, lacked the important contributors to viral pathogenesis: genes encoding proteins functioning in inhibition of cellular transcription and downregulation of the cellular antiviral response. To make these viruses incapable of transmission by mosquito vectors and to differentially regulate expression of viral structural proteins, their replication was made dependent on the internal ribosome entry sites, derived from other positive-polarity RNA (RNA ؉ ) viruses. The rational design of the genomes was complemented by selection procedures, which adapted viruses to replication in tissue culture and produced variants which (i) demonstrated different levels of replication and production of the individual structural proteins, (ii) efficiently induced the antiviral response in infected cells, (iii) were incapable of replication in cells of mosquito origin, and (iv) efficiently replicated in Vero cells. This modular approach to genome design is applicable for the construction of other alphaviruses with a programmed, irreversibly attenuated phenotype.The Alphavirus genus in the Togaviridae family contains almost 30 currently known members, which are distributed on all continents (48). Such a wide distribution implies evolution of the viruses in different, geographically isolated areas and their adaptation to different host and vector species. In mosquito vectors, alphaviruses cause a persistent, life-long infection that does not significantly interfere with the vectors' biological functions (59, 60). These viruses accumulate to high concentrations in mosquito salivary glands and are transmitted to vertebrate hosts during the blood meal. In vertebrates, alphaviruses develop an acute infection, characterized by a high-titer viremia, required for virus transmission to new mosquitoes during blood ingestion and continuation of the infection cycle. The high rates of alphavirus replication and viremia development are critically determined by efficient function of viral genome replication machinery and rapid accumulation of virus-specific structural proteins in the infected cells. However, another important contributor to the infectious process is the abilit...

Section: Resultssupporting

confidence: 92%

Design of Chimeric Alphaviruses with a Programmed, Attenuated, Cell Type-Restricted Phenotype

Kim

Foy

et al. 2011

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“…At 24 h postinfection, the subgenomic RNA was found at a concentration 3.5-fold higher than that of the genomic RNA, which was in agreement with results of previous studies (23). However, at days 6 to 8 postinfection, it was no longer detectable by the applied qPCR method.…”

Section: Resultssupporting

confidence: 91%

“…A theoretically plausible approach would be to develop and apply inefficiently replicating VEEV mutants having, for instance, modified RNA promoter elements. However, such modified viruses rapidly accumulate adaptive mutations in either promoter and/or ns polyproteins and readily evolve to the more efficiently replicating, cytopathic phenotype (23). In this study, we found a way around this problem by designing VEEV mutants having nuclear functions of capsid selectively inactivated by point mutations, without affecting the ability of the protein to package VEEV genomes and form infectious virions.…”

Section: Discussionmentioning

confidence: 99%

Interplay of Acute and Persistent Infections Caused by Venezuelan Equine Encephalitis Virus Encoding Mutated Capsid Protein

Krendelchtchikova

Liopo

et al. 2010

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Venezuelan equine encephalitis virus (VEEV) is a significant human and animal pathogen. The highlight of VEEV replication in vitro, in cells of vertebrate origin, is the rapid development of cytopathic effect (CPE), which is strongly dependent upon the expression of viral capsid protein. Besides being an integral part of virions, the latter protein is capable of (i) binding both the nuclear import and nuclear export receptors, (ii) accumulating in the nuclear pore complexes, (iii) inhibiting nucleocytoplasmic trafficking, and (iv) inhibiting transcription of cellular ribosomal and messenger RNAs. Using our knowledge of the mechanism of VEEV capsid protein function in these processes, we designed VEEV variants containing combinations of mutations in the capsid-coding sequences. These mutations made VEEV dramatically less cytopathic but had no effect on infectious virus production. In cell lines that have defects in type I interferon (IFN) signaling, the capsid mutants demonstrated very efficient persistent replication. In other cells, which have no defects in IFN production or signaling, the same mutants were capable of inducing a long-term antiviral state, downregulating virus replication to an almost undetectable level. However, ultimately, these cells also developed a persistent infection, characterized by continuous virus replication and beta IFN (IFN-␤) release. The results of this study demonstrate that the long-term cellular antiviral state is determined by the synergistic effects of type I IFN signaling and the antiviral reaction induced by replicating viral RNA and/or the expression of VEEV-specific proteins. The designed mutants represent an important model for studying the mechanisms of cell interference with VEEV replication and development of persistent infection.

“…The most probable explanation was that the mutations affected the efficiency of PS function. Of note, in our previous studies, this fragment did not function in the VEEV genome as part of any RNA promoters or enhancers (19,20,25), but was present in the helper RNAs, which were capable of self-packaging into virions (40).…”

Section: Resultsmentioning

confidence: 87%

Conservation of a Packaging Signal and the Viral Genome RNA Packaging Mechanism in Alphavirus Evolution

Kim

Firth

et al. 2011

Self Cite

104

103

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. Under natural conditions, these viruses are transmitted between vertebrate hosts by mosquito vectors. In mosquitoes, alphaviruses develop a persistent, life-long infection, characterized by the high concentrations of virus accumulated in the salivary glands (44), which promote virus transmission to amplifying hosts during the blood meal. In vertebrates, alphaviruses induce an acute infection and high-titer viremia, which is required for infection of new mosquitoes during blood ingestion. Thus, to support continuous virus circulation, this transmission cycle requires the presence of infectious virus at high titers in both vertebrates and invertebrates. To achieve this, alphaviruses have developed very efficient means of genome replication, structural protein synthesis, and interference with development of the antiviral response (2,3,13,14). These processes enable viral accumulation in both mosquitoes and the blood of infected vertebrate organisms.Alphaviruses enter the cells by receptor-mediated endocytosis, and after fusion of viral and endosomal membranes, nucleocapsids are released into the cytoplasm. Following their further disassembly, free viral genomes begin to serve as templates for translation of the nonstructural polyproteins (nsPs) and, later, for synthesis of the negative-strand RNA intermediates, forming the double-stranded RNAs (dsRNAs). The dsRNA-nsP complexes initially assemble at the plasma membrane (10, 15), and at later time points postinfection, some of them are transported into the cytoplasm on the surface of endosomes (11,15,38). These complexes synthesize large quantities of genomic and subgenomic RNAs. The latter RNA is efficiently translated into structural proteins that package genomic RNA into infectious virions. This RNA packaging process remains poorly understood. It is unclear how the viral genome is selectively packaged into viral particles, while the more abundant subgenomic RNA or cellular messenger RNAs and tRNAs are left behind. It is generally believed that such selective packaging is determined by the binding of capsid protein to particular sequences in the viral RNA termed the packaging signals (PSs) (45,46). In most of the alphaviruses, with the possible exception of Au...