Formation of nsP3-Specific Protein Complexes during Sindbis Virus Replication

Abstract

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.Alphaviruses are a widely distributed group of significant human and animal pathogens. Some of them, including Venezuelan, eastern, and western equine encephalitis viruses, cause serious febrile illness and encephalitis (26). Others cause diseases with mild symptoms that usually include rash, fever, and arthritis (19). Alphavirus structural and nonstructural proteins (nsPs) demonstrate an obvious homology, suggesting that replication of their genomes, interactions with host cell biology, and formation of viral particles have much in common (52).The alphavirus genome is a single-stranded RNA of positive polarity and almost 12 kb in length that mimics the structure of cellular mRNAs. It contains both a 5Ј methylguanylate cap and a 3Ј polyadenylate tail (27, 51). These features allow the translation of viral proteins by host cell machinery directly from the genome RNA. The 5Ј two-thirds of the genome is translated into nonstructural proteins that comprise the viral components of the replicative enzyme complex (RC) that is required for replication of the viral genome and transcription of the subgenomic RNA. The subgenomic RNA corresponds to the 3Ј third of the genome. It is synthesized from the subgenomic promoter and translated into viral structural proteins, which are dispensable for RNA replication. The RNAs...

The Amino-Terminal Domain of Alphavirus Capsid Protein Is Dispensable for Viral Particle Assembly but Regulates RNA Encapsidation through Cooperative Functions of Its Subdomains

“…However, in the last few years, our understanding of the alphavirus RNA replication mechanism has undergone some modifications. It was demonstrated that for some of the alphaviruses, such as SINV and VEEV, replication complexes are formed at the plasma membrane, and a major fraction of them remain there, even at later stages of the infection and continue to function as sites of viral RNA synthesis (49)(50)(51). Thus, RNA replication and its packaging into virions are likely to be more closely compartmentalized than we previously thought.…”

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells

“…In support of this idea, mass spectroscopy analyses of immunoprecipitated proteins from SINV-infected Rat-1 and BHK cells have identified time-dependent nsP3-associated recruitment of a variety of cellular proteins (Cristea et al, 2006;Frolova et al, 2006). These host proteins include Ras GTPase-activating protein SH3 domain-binding proteins (G3BP1 and G3BP2), 14-3-3 and heterogeneous nuclear ribonucleoproteins.…”

“…As other cellular proteins are recruited to SINV replication complexes (Cristea et al, 2006;Despres et al, 1995;Frolova et al, 2006;Gorchakov et al, 2008), PARP-1 might interact with these proteins and activate or inhibit them to promote SINV replication. It is also possible that PARP-1 is involved in stabilizing SINV replication complexes.…”

“…nsP3 associates with cellular membranes (Peranen & Kaariainen, 1991) and plays a role in regulating RNA synthesis (De et al, 1996;LaStarza et al, 1994b;Wang et al, 1994), but its exact function is unknown. nsP3 interacts with a variety of cellular proteins that are likely to play essential roles in the formation or function of virus replication complexes (Cristea et al, 2006;Despres et al, 1995;Frolova et al, 2006;Gorchakov et al, 2008), and a primary function for nsP3 may be to recruit the required cellular proteins to sites of SINV replication. However, the role of the different domains of nsP3 and their posttranslational modifications in recruitment of cellular proteins are unknown.…”

Controlled propagation of replication-competent Sindbis viral vector using suicide gene strategy

“…13 Figure 3a shows the design and construction of the RC vector with an integrated thymidine kinase gene isolated from herpes simplex virus (hsvtk) gene fusion at aa 389 of the nsp3 protein (RC-Sindbis/Nsp3-tk/Fluc). We first tested whether the vector proliferation can be controlled by systemic administration of the prodrug ganciclovir (GCV).…”