Role of Sindbis Virus Capsid Protein Region II in Nucleocapsid Core Assembly and Encapsidation of Genomic RNA

Frolova

et al. 2013

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein.The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

Section: Resultsmentioning

confidence: 76%

Venezuelan Equine Encephalitis Virus nsP2 Protein Regulates Packaging of the Viral Genome into Infectious Virions

Kim

Frolova

et al. 2013

“…Two copies of this sequence were present in the SINV DI RNA genome (26,28,39), and their deletion had a negative effect on RNA encapsidation, but not on RNA replication. Further studies strongly suggested that the interaction of this PS with SINV capsid is mediated by a short amino acid sequence located between amino acids (aa) 96 and 107 of the SINV capsid protein (29,43). These data provided a plausible explanation for specific packaging of SINV genomic RNA.…”

Section: Discussionmentioning

confidence: 98%

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

Kim

Firth

et al. 2011

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...

“…The carboxy terminus of the H68 peptide contains a short sequence of positively charged amino acids that might serve as an NLS. This sequence is a part of the large positively charged domain (aa 64 to 117 of the capsid) that is involved in encapsidation of viral genomic RNA (18,48) and contains a number of positively charged peptides, which, based on the computer prediction, may serve as NLS. Previously, we isolated a frameshift mutant of VEEV capsid protein, C VEE frsh ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin α/β That Obstructs Nuclear Pore Complex Function

Fish

Fornerod³

et al. 2010

104

139

Development of the cellular antiviral response requires nuclear translocation of multiple transcription factors and activation of a wide variety of cellular genes. To counteract the antiviral response, several viruses have developed an efficient means of inhibiting nucleocytoplasmic traffic. In this study, we demonstrate that the pathogenic strain of Venezuelan equine encephalitis virus (VEEV) has developed a unique mechanism of nuclear import inhibition. Its capsid protein forms a tetrameric complex with the nuclear export receptor CRM1 and the nuclear import receptor importin ␣/␤. This unusual complex accumulates in the center channel of the nuclear pores and blocks nuclear import mediated by different karyopherins. The inhibitory function of VEEV capsid protein is determined by a short 39-amino-acid-long peptide that contains both nuclear import and supraphysiological nuclear export signals. Mutations in these signals or in the linker peptide attenuate or completely abolish capsid-specific inhibition of nuclear traffic. The less pathogenic VEEV strains contain a wide variety of mutations in this peptide that affect its inhibitory function in nuclear import. Thus, these mutations appear to be the determinants of this attenuated phenotype. This novel mechanism of inhibiting nuclear transport also shows that the nuclear pore complex is vulnerable to unusual cargo receptor complexes and sheds light on the importance of finely adjusted karyopherin-nucleoporin interactions for efficient cargo translocation.