Previously, we showed that the cellular protein PCBP2 interacts with the VSV P protein and inhibits viral replication at the level of gene expression (23
). In the present work, we conducted studies to understand how PCBP2 inhibits VSV replication. The rationale for these studies was derived from the observations that (i) PCBP2 is a component of cellular SGs and PBs (2
); (ii) PCBP2 interacts and colocalizes with TIA1 in SGs (22
); and (iii) several viruses have been shown to induce and/or interfere with the formation of TIA1/TIAR-containing SGs (5
). We show here that TIA1, but not TIAR, inhibits VSV growth at the level of viral gene expression and/or replication. Following VSV infection, TIA1 translocates from the nucleus to the cytoplasm and aggregates into cytoplasmic granules similar to the SGs seen in the cytoplasm of cells under stress conditions. These granules also contain the viral replication proteins and RNA as well as PCBP2. The formation of these granules required viral replication and protein synthesis but was independent of cellular microtubule and microfilament networks or cellular transcription. The VSV-induced granules, termed “SG-like structures,” appear to be similar to those described previously as cytoplasmic granules or transcription and replication inclusions (27
). However, these studies could not unequivocally rule out the possibility that viral RNA synthesis occurred in the cytoplasm and some of the newly synthesized RNAs quickly accumulated at these sites. Therefore, the question of whether the cytoplasmic granules or inclusions represent the sites of viral RNA synthesis remains unresolved. The formation of the inclusions may reflect the host cell's response to VSV infection (28
). This cellular response may depend on the synthesis of critical levels of viral proteins and/or RNA and their interactions with host cell components. Although the host cell proteins in these granules have not been identified, it is tempting to speculate that the granules are similar to the SG-like structures described here and may represent aggregation of translational silencing as well as RNA-binding proteins, such as TIA1 and PCBP2, that are involved in inhibiting viral gene expression. Since both TIA1 and TIAR are apoptotic proteins (47
), it is likely that sequestration of these molecules in SG-like structures in infected cells positively impacts cell survival and virus replication.
Although the induction of SG-like structures in infected cells required viral replication and protein synthesis, our inability to detect such structures (data not shown) in cells infected with VSV defective interfering particle-infected cells constitutively expressing the viral replication proteins (49
) suggests that viral transcription and/or threshold concentrations of the viral proteins and RNAs are required for their formation. This interpretation is consistent with the delayed appearance (3 hpi) of the structures in infected cells, even though virus transcription/replication and protein synthesis can be detected within the first hour of infection (unpublished data).
Numerous viruses have been shown to either induce (11
) or suppress (6
) SG formation in infected cells. It appears that the induction or suppression of SGs and of each of their component proteins plays divergent roles in either facilitating or inhibiting virus infections. Infection with poliovirus results in early induction of SGs (15
) that are also maintained at later times of infection with compositionally different cellular SG proteins (15
). Interestingly, the compositionally different SGs that appear late in poliovirus infection, termed “pseudo-SGs,” contain mostly self-aggregated TIA1 and lack many of the canonical SG components (20
). A mutant vaccinia virus lacking the double-stranded RNA-binding E3L protein induces cytoplasmic antiviral granules (AVGs) that inhibit virus replication (17
). These AVGs, which are not generated in cells infected with wt vaccinia virus, contain TIA1, G3BP, and other RNA-binding proteins and restrict the replication of the ΔE3L mutant virus (17
). On the other hand, hepatitis C virus (HCV)-induced SGs contain not only factors required for HCV replication (Rck1) but also antiviral factors (PCBP2, G3BP1, TIAR, Xrn1) (52
). Thus, whether the SGs, AVGs, or pseudo-SGs are generated by the host cells in response to virus infection or are generated by the viral proteins and RNAs to facilitate or limit virus infection remains an open question at this time. Nevertheless, it appears that specific interactions between viral and cellular components over multiple pathways through the recruitment of common and unique SG markers may be crucial for the formation of these granular structures.
In the case of VSV, TIA1/TIAR and PCBP2, along with the viral proteins and RNAs, were found to be localized to the SG-like structures, whose induction was independent of the cellular microtubule or microfilament network. Unlike canonical SGs, whose formation is dependent not only on the cytoskeletal network but also on cellular transcription, the SG-like structures in VSV-infected cells formed in the absence of cellular transcription and were independent of the cytoskeleton. Additional studies will be necessary to determine the mechanism of formation of the SG-like structures in VSV-infected cells. VSV infection also did not interfere with the formation of canonical SGs containing eIF3 or eIF4A induced by SA treatment, suggesting that the SG-like structures induced in VSV-infected cells may have a unique cellular protein composition. Further work to identify the cellular proteins that accumulate in these SG-like structures will be important to determine the role played by these structures in the VSV infection cycle. The observations that (i) the SG-like structures are distinct from canonical SGs, (ii) the TIA1 protein, which blocks the translation of cellular mRNA in canonical SGs, did not inhibit VSV mRNA translation in infected cells (), and (iii) VSV RNA synthesis is enhanced in TIA1-depleted cells suggest that TIA1 downregulates VSV infection through a mechanism other than translational suppression, its traditional role.
The involvement of eIF2α phosphorylation and the double-stranded RNA-dependent protein kinase (protein kinase R [PKR]) in the formation of VSV-induced SG-like structures has not been examined directly, although the level of SG-like structures increased concomitantly with the level of eIF2α phosphorylation. It will be of interest to explore whether PKR, which is activated in VSV infection and restricts virus replication (53
), or other eIF2α-specific kinases play any role in the induction of the SG-like structures.
The observations that VSV replication is enhanced in cells when TIA1 is depleted transiently (by siRNA treatment) () but is inhibited in cells permanently depleted of TIA1 (knockout MEFs) () appear paradoxical. However, since TIA1 is involved in cellular gene expression, including translational suppression (29
) and mRNA processing (54
), it is possible that the expression of a certain cellular gene(s) required for VSV growth may have been permanently compromised in MEFs derived from the knockout mouse. Alternatively, TIA1 depletion in MEFs may have resulted in the activation of an antiviral gene(s) to block VSV growth. In contrast to the knockout of TIA1 in MEFs, transient depletion of TIA1 in HeLa cells may not have permanently affected these cellular factors. Therefore, the true effects of TIA1 on VSV growth could be discerned in transient depletion experiments, whereas in cells stably depleted of the protein, the combined effects of TIA1 depletion and its subsequent effects on cellular proteins are observed. Such a contention is supported by the fact that siRNA-mediated depletion of TIA1 from wt MEFs resulted in increased VSV growth whereas ectopic expression of the protein in TIA1-knockout MEFs led to inhibition of VSV growth. Proteomic and transcriptomic analysis of wt and TIA1-knockout MEFs may reveal important clues for a better understanding of the role of TIA1 in VSV infection.
In summary, our studies presented here reveal that TIA1 inhibits VSV replication and colocalizes with PCBP2, another cellular protein shown previously (23
) to inhibit VSV replication, to form SG-like structures in infected cells. Studies to determine the cellular protein composition of these SG-like structures and their roles in VSV-infected cells are ongoing.