Evidence for Translational Regulation by the Herpes Simplex Virus Virion Host Shutoff Protein

The herpes simplex virus (HSV) virion host shutoff protein (vhs) encoded by gene UL41 is an mRNA-specific RNase that triggers accelerated degradation of host and viral mRNAs in infected cells. We report here that vhs is also able to modulate reporter gene expression without greatly altering the levels of the target mRNA in transient-transfection assays conducted in HeLa cells. We monitored the effects of vhs on a panel of bicistronic reporter constructs bearing a variety of internal ribosome entry sites (IRESs) located between two test cistrons. As expected, vhs inhibited the expression of the 5′ cistrons of all of these constructs; however, the response of the 3′ cistron varied with the IRES: expression driven from the wild-type EMCV IRES was strongly suppressed, while expression controlled by a mutant EMCV IRES and the cellular ApaF1, BiP, and DAP5 IRES elements was strongly activated. In addition, several HSV type 1 (HSV-1) 5′ untranslated region (5′ UTR) sequences also served as positive vhs response elements in this assay. IRES activation was also observed in 293 and HepG2 cells, but no such response was observed in Vero cells. Mutational analysis has yet to uncouple the ability of vhs to activate 3′ cistron expression from its shutoff activity. Remarkably, repression of 5′ cistron expression could be observed under conditions where the levels of the reporter RNA were not correspondingly reduced. These data provide strong evidence that vhs can modulate gene expression at the level of translation and that it is able to activate cap-independent translation through specific cis-acting elements.The virion host shutoff protein (vhs) encoded by herpes simplex virus (HSV) gene UL41 is an endoribonuclease that is packaged into the tegument of mature HSV virions. Once delivered into the cytoplasm of newly infected cells, vhs triggers shutoff of host protein synthesis, disruption of preexisting polysomes, and degradation of host mRNAs (reviewed in reference 62). The vhs-dependent shutoff system destabilizes many cellular and viral mRNAs (36, 46, 67). The rapid decline in host mRNA levels presumably helps viral mRNAs gain access to the cellular translational apparatus. In addition, the relatively short half-lives of viral mRNAs contribute to the sharp transitions between the successive phases of viral protein synthesis by tightly coupling changes in the rates of synthesis of viral mRNAs to altered mRNA levels (46). These effects enhance virus replication and may account for the modest reduction in virus yield displayed by vhs mutants in cultured Vero cells (55, 61).vhs also plays a critical role in HSV pathogenesis: vhs mutants are severely impaired for replication in the corneas and central nervous systems of mice and cannot efficiently establish or reactivate from latency (63, 65, 66). Mounting evidence indicates that this attenuation stems at least in part from an impaired ability to disarm elements of the innate and adaptive host immune responses (reviewed in reference 62). For example, vhs suppresses certain innate cellular antiviral responses, including production of proinflammatory cytokines and chemokines (68); dampens the type I interferon system (11, 45, 49, 78); and blocks activation of dendritic cells (58). Moreover, vhs mutants display enhanced virulence in knockout mice lacking type I interferon (IFN) receptors (37, 45) or Stat1 (48) and are hypersensitive to the antiviral effects of IFN in some cells in tissue culture (11, 49, 68). Thus, vhs is arguably a bona fide virulence factor.vhs present in extracts of HSV virions or purified from bacteria has nonspecific RNase activity capable of degrading all RNA substrates (15, 70, 71, 79). However, vhs is highly selective in vivo, targeting mRNAs and sparing other cytoplasmic RNAs (36, 46). In vivo and in mammalian whole-cell extracts, vhs-induced decay of at least some mRNAs initiates near regions of translation initiation and proceeds in an overall 5′-to-3′ direction (12, 13, 29, 52). Moreover, vhs binds to the translation initiation factors eIF4H, eIF4B, and eIF4A II, all components of the cap recognition factor eIF4F (10, 16, 17). Thus, it has been proposed that vhs selectively targets actively translated mRNAs through interactions with eIF4F components (17). Consistent with this hypothesis, recent data document that eIF4H is required for vhs activity in vivo (59).A previous report from this laboratory documented that the internal ribosome entry sites (IRESs) of the picornaviruses poliovirus and encephalomyocarditis virus (EMCV) strongly target vhs-induced RNA cleavage events to sequences immediately 3′ to the IRES in an in vitro translation system derived from rabbit reticulocyte lysates (RRL) (13). IRES elements are highly structured RNA sequences that are able to direct cap-independent translational initiation (reviewed in references 21, 25, 30, and 64). In the case of the poliovirus and EMCV elements, this is achieved by directly recruiting the eIF4F scaffolding protein eIF4G, thus bypassing the requirement for the cap-binding eIF4F subunit, eIF4E (reviewed in reference 30). Based on these data, we suggested that vhs is strongly targeted to the picornavirus IRES elements via interactions with eIF4 factors.A growing number of cellular mRNAs have been proposed to bear IRES elements in their 5′ untranslated regions (5′ UTRs). These include many that are involved in cellular stress responses, apoptosis, and cell cycle progression (24, 64, 74). Given the striking ability of picornavirus IRES elements to target vhs RNase activity in vitro, we asked whether viral and cellular IRES elements are able to modify the susceptibility of mRNAs to vhs in vivo. During the course of preliminary experiments designed to test this hypothesis, we unexpectedly discovered that vhs is able to strongly activate gene expression controlled by some cellular IRES elements and HSV 5′ UTR sequences in in vivo bicistronic reporter assays. These observations are the subject of the present report.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.