In this study we have isolated and identified 29 host factors that associate with nsP4, the viral RNA-dependent RNA polymerase, at early (6 h) and late (12 h) times of infection with SINV. The use of a SINV expressing 3×Flag epitope-tagged nsP4 enabled the identification of host factors most likely to directly influence RNA replication and transcription. Some of the 29 proteins we found associated with nsP4 may be important for the SINV life cycle and in the present study we identified G3BP1 and G3BP2 as playing a role in the SINV replication process. Control isolations using cells infected with a virus expressing GFP containing a 3×Flag epitope tag (TE/5′2J/GFP-3×Flag) were used to distinguish nonspecific association to the Flag tag, anti-Flag antibodies, or magnetic beads. However, during incubation of the cell lysate with magnetic beads, isolated nsP4 complexes can bind to additional nonspecific proteins. Although we utilized a relatively rapid (1-h) affinity isolation incubation time to minimize nonspecific interactions (7
), it is unlikely that all of the identified nsP4-interacting factors are specifically associated with nsP4. In addition to proteins isolated in control samples (marked as likely contaminants in Table and Table S1 in the supplemental material), other potential nonspecifically interacting proteins can be identified based on previous experience and the literature. Highly abundant cytoskeletal (tubulin) or ribosomal proteins have been noted as possible contaminants in Flag affinity isolations (4
), and we previously identified other proteins (glyceraldeyde-3-phosphate dehydrogenase, prohibitin, and fibrillarin [data not shown]) in control affinity isolations from unrelated experiments using the Flag epitope tag. Thus, most of the proteins identified in the current study should be considered as potential factors that could be important for SINV replication and its regulation. In the present study we show that G3BP1 and G3BP2 play roles in SINV replication; additional studies are required to confirm the other interactions and elucidate which factors are important for SINV replication.
Five host factors—UNC84, PRMT5, GNB2L1, SFRS10, and ANXA1—were found to be associated with nsP4 only at 6 hpi, not being observed in complexes isolated at 12 h. Aside from GNB2L1, these proteins were not previously detected in affinity isolations of nsP2 (1
) or nsP3 (6
). Since both nsP2 and nsP3 are present in early replication complexes, the failure to identify UNC84, PRMT5, SFRS10, and ANXA1 in previous studies is rather surprising. However, given the excess of P123 synthesis compared to nsP4 in SINV-infected cells, it is possible that, in previous studies, the host factors interacting with the replicase-associated nsP2 and nsP3 were obscured by host factors associated with the relatively larger amounts of nsP2 and nsP3 residing outside of replication complexes. This in fact was a major consideration that led us to utilize a tagged nsP4 for the isolation of host factors possibly involved in viral RNA replication. The finding of five factors in association with nsP4 only at early times after infection suggests their involvement in early, but not late RNA replication processes. For example, these factors might aid the formation of viral replication complexes, facilitate minus-strand synthesis or mediate the switch from minus to plus strand synthesis. Interestingly, annexins are involved in membrane reorganization (reviewed in reference 15
) and UNC84 (also known as SUN2) is a nuclear membrane protein proposed to be involved in positioning of the nucleus of muscle cells through protein-protein interactions (24
). One can speculate that these proteins may be involved in mediating the proper membrane topology and localization to facilitate the formation of the SINV replication complexes, which are found in association with host membranes (13
). PRMT5 (also found only at 6 h) is known to associate with MEP50 (found at both 6 and 12 h) as components of the methylosome, which modifies arginine residues in Sm splicosome proteins to dimethylarginine, facilitating their recruitment to the survival of motor neurons complex and the formation of small nuclear ribonucleoprotein particles (9
). Although one can conjecture a role for arginine methylation of host or viral proteins in the formation of SINV replication complexes, this protein modification has not been studied in the context of SINV infection. Another protein identified only at 6 h, SFRS10, also known as Tra2, is involved in splicing regulation (33
) and might participate in replicase formation or the regulation of RNA replication through its RNA binding activity. However, in our experience splicing factors are common contaminants in Flag affinity isolations (data not shown). In addition, MEP50 and PRMT5 have been noted as common contaminants in Flag affinity isolations (4
). Therefore, in future studies it will be important to confirm the specificity of these interactions.
Ten proteins were found associated with nsP4 only at late times (12 h) after infection. Although the number of proteins identified precludes discussing their individual potential roles in SINV replication, we speculate that some of these proteins may play roles in later events of the replication process, possibly including regulation of assembly processes, such as diverting genomic RNA toward assembly rather than translation. Since SINV infection of most vertebrate cells results in severe alterations in morphology, cytopathic effect, and death by apoptosis (26
), a role for these factors in regulating the morphological changes and cell death caused by SINV could also be postulated. Of the factors isolated only at 12 h, the two ribosomal proteins were also identified as associating within nsP3-containing complexes, while the other eight were not previously identified in association with nsP2 (1
) or nsP3 (6
). Determination of the functions of these proteins in SINV replication will require additional study.
The factors found at both early and late times are candidates for host factors important for minus and plus strand RNA synthesis, as well as subgenomic RNA transcription. Interestingly, 14 host factors were found in association with nsP4 at both early and late times after infection. These included G3BP1 and G3BP2, which were previously identified in nsP3-containing complexes (6
), as well as in association with nsP2 (1
). We also identified multiple 14-3-3 proteins in association with nsP4 at both early and late times. 14-3-3 proteins have also previously been found in association with nsP3 (6
). In our previous study (6
) several 14-3-3 isoforms were found associated with nsP3 after 6 h of infection and longer but not at times earlier than 6 h (6
). Six proteins—MEP50, HSP90, TRIM30, 14-3-3θ, TYMS, and SERPINH1—found associated with nsP4 at both 6 and 12 h after infection were not previously identified in complexes isolated by immunoprecipitation of nsP2 or nsP3.
In the present study we chose to follow up on a possible functional role for G3BP1 and G3BP2 in SINV replication, since these proteins were previously detected in association with other SINV nsPs and demonstrated a robust association with nsP4 (see Coomassie blue-stained bands, Fig. ). Our data suggest that G3BP1 and G3BP2 function to limit SINV polyprotein expression. Limiting SINV polyprotein expression could be beneficial to virus replication or could represent a host cell response to limit virus replication. Although silencing of the G3BPs had minimal effects on virion production in cell culture (Fig. ), it is possible that the recruitment of G3BP1 and G3BP2 is critical for regulating SINV replication in vivo
, for example, in specific cell types, and may play a role in the pathogenesis of this virus. G3BP1 assembles stress granules (40
), sites of mRNA triage for continued translation or degradation (21
). As has been suggested by others (17
), SINV sequestration of G3BP1 may serve to interfere with the host cell stress response. Interestingly, poliovirus, another positive-strand RNA virus, cleaves G3BP1 with a resultant interference in stress granule formation (41
). G3BP1 has also been found in association with replication complexes of the RNA virus hepatitis C; however, in this case G3BP1 knock down was associated with decreased RNA replication (43
). G3BP2 has been implicated in control of NF-κB signaling by retaining IκB/NF-κB complexes in the cytoplasm (30
). Thus, the recruitment of G3BP2 into nsP4-containing complexes might serve to limit G3BP2's effects on NF-κB signaling.
We demonstrate here, using a luciferase-expressing SINV, that silencing of G3BP1 and G3BP2 results in increased polyprotein expression from the viral genomic RNA (Fig. ). This effect could be direct or, alternatively, could be due to effects on viral RNA replication. Although we detected a trend toward increased polyprotein expression using a temperature-sensitive virus defective in RNA replication (Fig. ), we were unable to reproducibly detect significantly increased luciferase activity. We found no evidence of leaky replication of the temperature-sensitive mutant (Fig. ) to account for the occasional statistically significant increase in luciferase activity. Although the lack of a reproducible significant effect on replication incompetent SINV RNA translation suggested that the G3BPs might directly affect RNA replication, we also did not detect significantly increased RNA levels upon G3BP silencing. It is possible that reduction of G3BP levels results in a subtle effect on polyprotein expression, which, upon replication of the viral RNA gets amplified to statistically significant levels as the new RNA templates enter the translational pool. Interestingly, upon silencing of the G3BPs, a 3- to 5-fold enhancement of polyprotein expression from replication competent virus was maintained over time after infection (Fig. ) but did not result in substantial increases in viral RNA (Fig. ) or virion production (Fig. ). We interpret the small increases in viral RNA levels and virion production seen upon G3BP silencing to be a result of the enhanced genome polyprotein expression. However, a small direct effect on RNA replication or virion production cannot be excluded, since experiments to measure RNA replication and virion production independently from polyprotein expression are not possible.
Given the RNA binding activity of the G3BPs and their role in the formation of stress granules, sites where mRNA translation and degradation are regulated (21
), our results suggest that G3BP1 and G3BP2 might normally function to limit SINV genome translation by recruitment of the viral RNA into the stress granule pathway. It is somewhat surprising that reducing the level of these proteins did not have a more dramatic effect. One possible explanation for the modest effect we detected (3- to 8-fold) is that the recruitment of G3BPs by nsPs that are not directly involved in RNA replication results in a functional depletion of G3BPs to which our siRNA-mediated silencing adds little. The fact that we can demonstrate an effect highlights a possible role for the stress granule pathway in the host cell response to SINV infection. Alternatively, it is possible that a subtle reduction in polyprotein expression is beneficial for the virus, in which case SINV may have usurped this pathway to its advantage. An alternative possibility is that the G3BPs recruit SINV RNA out of the translating pool of RNA and into nsP4-containing replication complexes. Interestingly, since G3BP1 was identified as a helicase capable of unwinding both DNA and RNA substrates (5
), a role in clearing the viral RNA of proteins in preparation for the switch from translation to replication is possible. Although this would predict a delay or reduction in the formation of replication complexes in G3BP silenced cells, any newly replicated RNA that is produced would be predicted to produce more nsPs due to enhanced time in the translating pool. Understanding whether the G3BPs play a role in replication template recruitment and/or more direct inhibitory roles on translation or polyprotein stability requires further investigation.
In the present study we successfully generated a viable SINV expressing an epitope-tagged version of the viral RNA-dependent RNA polymerase (nsP4). Affinity isolation of the tagged nsP4 led to the identification of 29 proteins that were associated within the nsP4-containing complexes. While testing for which of these host proteins play active roles in SINV RNA replication and understanding the details of the host factor-nsP4 interaction require further investigation, knowledge in this area could lead to new approaches to disrupt these critical interactions and limit the devastating diseases caused by alphaviruses.