Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Abstract

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.The Alphavirus genus contains over 25 recognized viruses with a wide geographic distribution. Venezuelan equine encephalitis virus (VEE) is a New World alphavirus that is maintained in nature by cycling between a mosquito vector and susceptible vertebrate hosts. VEE is responsible for periodic outbreaks of disease in humans and equines and is classified as a select agent, making it a prominent pathogen among the alphaviruses.Alphaviruses possess a genome of single-stranded messagesense RNA that is approximately 11.5 kb in length. The alphavirus genome is organized such that the 5Ј two-thirds of the genome encodes four nonstructural proteins (nsP1 through nsP4), whereas the 3Ј one-third encodes three mature structural proteins (capsid, E2, and E1) that are expressed at high levels after transcription from an internal 26S subgenomic mRNA promoter. The viral genome appears to be similar to cellular mRNAs, since it contains 5Ј and 3Ј untranslated regions, with a 5Ј-terminal methylguanylate cap and a 3Ј-terminal polyadenylate tail. Upon release into a host cell, the viral genomic RNA is directly translated by the cellular translation machinery. The nonstructural proteins are synthesized as two polyproteins, termed P123 and P1234. P123 results when translation terminates at an opal termination codon between nsP3 and nsP4. When t...

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

“…Among identified interactions, CHIKV nsP2 was found to bind heterogeneous nuclear hnRNP-K and vimentin (VIM), in agreement with previous reports showing that both cellular proteins copurified with SINV nsP2 (3,7). We also confirmed that nsP2 interacts with 40S ribosomal protein S6 (RpS6), polyadenylate-binding protein (PABPC1), actin (ACTB), and elongation factor 1 (EEF1A1) (3,37), but these interactions were only supported by single positive yeast colonies and thus discarded from the high-confidence interaction data set. All other identified interactions are novel and were not previously reported in the literature for any alphavirus.…”

“…Members of the hnRNP family are involved in RNA processing, trafficking, and translation and have been found associated with alphavirus genomic and/or subgenomic RNA molecules, probably contributing to their synthesis and translation. Interactions between alphavirus nsP2 proteins and ribosomal components, in particular RpS6, were also reported by Montgomery et al (37) and likely contribute to the hijacking of translational machinery by viral factors. In addition to their different roles as cofactors of the viral polymerase complex, nsP2 proteins from Old World alphaviruses have been described as virulence factors responsible for the transcriptional and translational shutoff observed in infected host cells and the inhibition of interferon (IFN)-mediated antiviral responses (6, 14, 16-19, 21, 33, 49).…”

“…Cellular interactors of CHIKV identified by HT-Y2H were analyzed in the context of interaction data already available in the literature for other Old World alphaviruses. Several groups have already identified cellular interactors of nsP2, nsP3, and nsP4 for SINV, in particular, by using recombinant viruses expressing tagged viral proteins that allowed the affinity purification and mass spectrometry-based identification of cellular partners (3,7,10,11,15,22,37). Interaction data reported in these manuscripts were gathered and reformatted using identical protein ID numbers (see Table S1 in the supplemental material) and collapsed with our HT-Y2H data to build a map of cellular factors targeted by nonstructural proteins of Old World alphaviruses (Fig.…”

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

“…The direct or indirect interactions of nsP2 and CP have been experimentally confirmed, at least for VEEV and SINV. CPs of both viruses are very efficiently coprecipitated with nsP2 from virus-infected cells (27), and both nsP2 and capsid proteins are associated with ribosomes in coimmunoprecipitation and polysome fractionation experiments (35)(36)(37).…”

Venezuelan Equine Encephalitis Virus Disrupts STAT1 Signaling by Distinct Mechanisms Independent of Host Shutoff

“…Nuclear purification was performed as previously described (35), except the crude nuclear fraction was not banded, but rather was washed five consecutive times in 0.5 to 1.0 ml of homogenization medium prior to lysis in nuclear extraction buffer. Equal amounts of total protein from cytoplasmic and nuclear extracts were analyzed by Western blot as described above, and the purity of the nuclear extracts was determined by Western blotting to detect GRP78, which localizes to the endoplasmic reticulum and should not be found within nuclear fractions.…”