Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However The Alphavirus genus of the Togaviridae family contains almost 30 currently known members, which are distributed all over the world and are grouped into eight serocomplexes (57). Alphaviruses cause a variety of diseases ranging from mild rash and arthritis to a serious febrile illness and encephalitis (27) that may result in death and severe neurological disorders (7,21). In natural conditions, alphaviruses are transmitted between vertebrate hosts by mosquito vectors. In mosquitoes, they develop a persistent, life-long infection (4) characterized by the presence of infectious virus in the salivary glands, facilitating the infection of avian or mammalian hosts during the mosquito's blood meal. In infected vertebrate hosts, alphaviruses induce an acute infection, which leads to the high-titer viremia required for infection of new mosquitoes during blood ingestion. Replication in cultured cells mirrors the natural transmission cycle: alphaviruses develop a highly productive, cytopathic infection in cells of vertebrate origin, characterized by numerous modifications of the intracellular environment and cell death within 24 to 48 h postinfection (55). In mosquito cells, they develop a noncytopathic, persistent or chronic infection that also results in high titer virus release.Thus, alphaviruses are capable of efficient replication in fundamentally different types of cells (of insect and vertebrate origin) and appear to utilize a number of diverse host protein factors required for numerous processes in viral replication. Most importantly, in order to develop efficient spreading of infection in both cell types, they have to interfere with two different antiviral systems. In insect cells, the antiviral effect is determined mostly by double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) (47). On the other hand, in vertebrate cells, the antiviral response is characterized by the induction of hundreds of cellular genes and is activated by pattern recognition receptors (PRRs), which detect virus-specific dsRNA molecules, synthesized during virus replication, and other virus-specific products and processes (29).The alphavirus genome is represented by a single-stranded RNA of positive polarity that is almost 11.5 kb in length (53,54). It mimics the structure of cellular mRNAs, in that it contains a cap at the 5Ј terminus and a poly(A) tail at the 3Ј terminus. This RNA contains two open reading fr...

The innate immune response is the first line of defense against most viral infections. Its activation promotes cell signaling, which reduces virus replication in infected cells and leads to induction of the antiviral state in yet-uninfected cells. This inhibition of virus replication is a result of the activation of a very broad spectrum of specific cellular genes, with each of their products usually making a small but detectable contribution to the overall antiviral state. The lack of a strong, dominant function for each gene product and the ability of many viruses to interfere with the development of the antiviral response strongly complicate identification of the antiviral activity of the activated individual cellular genes. However, we have previously developed and applied a new experimental system which allows us to define a critical function of some members of the poly(ADP-ribose) polymerase (PARP) family in clearance of Venezuelan equine encephalitis virus mutants from infected cells. In this new study, we demonstrate that PARP7, PARP10, and the long isoform of PARP12 (PARP12L) function as important and very potent regulators of cellular translation and virus replication. The translation inhibition and antiviral effect of PARP12L appear to be mediated by more than one protein function and are a result of its direct binding to polysomes, complex formation with cellular RNAs (which is determined by both putative RNA-binding and PARP domains), and catalytic activity. IMPORTANCEThe results of this study demonstrate that interferon-stimulated gene products PARP7, PARP10, and PARP12L are potent inhibitors of the replication of Venezuelan equine encephalitis virus and other alphaviruses. The inhibitory functions are determined by more than a single mechanism, and one of them is based on the ability of these proteins to regulate cellular translation. Interference with the cellular translational machinery depends on the integrity of both the amino-terminal domain, containing a number of putative RNA-binding motifs, and the catalytic function of the carboxy-terminal PARP domain. The PARP-induced changes in translation efficiency appear to have a more potent effect on the synthesis of virus-specific proteins than on that of cellular proteins, thus making PARP-specific translational downregulation an important contributor to the overall development of the antiviral response.

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...

Alphaviruses represent a highly important group of human and animal pathogens, which are transmitted by mosquito vectors between vertebrate hosts. The hallmark of alphavirus infection in vertebrates is the induction of a high-titer viremia, which is strongly dependent on the ability of the virus to interfere with host antiviral responses on both cellular and organismal levels. The identification of cellular factors, which are critical in orchestrating virus clearance without the development of cytopathic effect, may prove crucial in the design of new and highly effective antiviral treatments. To address this issue, we have developed a noncytopathic Venezuelan equine encephalitis virus (VEEV) mutant that can persistently replicate in cells defective in type I interferon (IFN) production or signaling but is cleared from IFN signaling-competent cells. Using this mutant, we analyzed (i) the spectrum of cellular genes activated by virus replication in the persistently infected cells and (ii) the spectrum of genes activated during noncytopathic virus clearance. By applying microarray-based technology and bioinformatic analysis, we identified a number of IFN-stimulated genes (ISGs) specifically activated during VEEV clearance. One of these gene products, the long isoform of PARP12 (PARP12L), demonstrated an inhibitory effect on the replication of VEEV, as well as other alphaviruses and several different types of other RNA viruses. Additionally, overexpression of two other members of the PARP gene superfamily was also shown to be capable of inhibiting VEEV replication.

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.