Solubilization and immunoprecipitation of alphavirus replication complexes.

Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsP1, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of approximately 120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsP1, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.     

Changes of the secondary structure of the 5' end of the Sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations

Both the 5' end of the Sindbis virus (SIN) genome and its complement in the 3' end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5' untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5' sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5' UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5' UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.     

Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci

Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response.     

Molecular defects caused by temperature-sensitive mutations in Semliki Forest virus nsP1

Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.     

RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells

RNA interference (RNAi) has recently shown promise as a mode of inhibition of slowly replicating viruses causing chronic diseases such as hepatitis C. To investigate whether RNAi is also feasible for rapidly growing RNA viruses such as alphaviruses, we tested the ability of expressed short hairpin RNAs (shRNAs) to inhibit the Semliki Forest virus (SFV), a rapidly replicating positive-strand RNA virus. Plasmids expressing shRNAs targeting SFV target sequences under the control of a human U6 promoter were introduced into BHK-21 cells. The targets included sequences encoding nonstructural (nsP1, 2, and 4) and structural (capsid) proteins as well as nonviral sequences serving as control targets. Twenty-four to 48 hours following transfection with shRNA plasmids, the cells were infected with replication-competent or replication-deficient recombinant SFV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) of approximately 5. Viral replication was monitored by fluorescence microscopy and flow cytometry. Specific and marked reduction of viral replication was observed with shRNAs targeting nsP1 and nsP4. The degree of inhibition of the replication-deficient SFV was >or=70% over a 5-day period, a level similar to the transfection efficiency, suggesting complete inhibition of nonreplicating virus in the transfected cell population. However, only nsP1 shRNA was inhibitory against replication-competent SFV (approximately 30%-50% reduction), and this effect was transient. No inhibition was observed with control shRNAs. In contrast to the recent success of RNAi approaches for slowly growing viruses, these results illustrate the challenge of inhibiting very rapidly replicating RNA viruses by RNAi. However, the addition of RNAi approaches to other antiviral modalities might improve the response to acute infections.     

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.     

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Different types of nsP3-containing protein complexes in Sindbis virus-infected cells

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.     

Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.     

A new role for ns polyprotein cleavage in Sindbis virus replication

One of the distinguishing features of the alphaviruses is a sequential processing of the nonstructural polyproteins P1234 and P123. In the early stages of the infection, the complex of P123+nsP4 forms the primary replication complexes (RCs) that function in negative-strand RNA synthesis. The following processing steps make nsP1+P23+nsP4, and later nsP1+nsP2+nsP3+nsP4. The latter mature complex is active in positive-strand RNA synthesis but can no longer produce negative strands. However, the regulation of negative- and positive-strand RNA synthesis apparently is not the only function of ns polyprotein processing. In this study, we developed Sindbis virus mutants that were incapable of either P23 or P123 cleavage. Both mutants replicated in BHK-21 cells to levels comparable to those of the cleavage-competent virus. They continuously produced negative-strand RNA, but its synthesis was blocked by the translation inhibitor cycloheximide. Thus, after negative-strand synthesis, the ns proteins appeared to irreversibly change conformation and formed mature RCs, in spite of the lack of ns polyprotein cleavage. However, in the cells having no defects in alpha/beta interferon (IFN-alpha/beta) production and signaling, the cleavage-deficient viruses induced a high level of type I IFN and were incapable of causing the spread of infection. Moreover, the P123-cleavage-deficient virus was readily eliminated, even from the already infected cells. We speculate that this inability of the viruses with unprocessed polyprotein to productively replicate in the IFN-competent cells and in the cells of mosquito origin was an additional, important factor in ns polyprotein cleavage development. In the case of the Old World alphaviruses, it leads to the release of nsP2 protein, which plays a critical role in inhibiting the cellular antiviral response.     

Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages

Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.     

Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.