The P protein of VSV is the noncatalytic subunit of the viral RdRp. Its phosphorylation status and interactions with the other viral proteins involved in viral genome transcription and replication regulate its activity in these processes (20
). Furthermore, P has been shown to play a role in assembly of infectious virus particles (19
). Since the P protein regulates multiple functions in the life cycle of VSV, we reasoned that it must interact with many host cell proteins. Currently, little is known about the host cell proteins that interact and modulate functions of the P proteins of VSV. One early report suggested that the P protein associates with c-src (6
); however, the functional significance of this association is unclear. Interaction of the P protein of rabies virus with dynein light chain LC8 has been shown to be required for its activity in viral genome transcription and replication (52
). The P proteins of rabies virus and Borna disease virus have also been shown to antagonize the IFN response (13
). The Borna disease virus P protein interacts with GABARAP, and this interaction results in relocalization of GABARAP from the cytoplasm to nucleus (50
). The nucleocapsid proteins of negative-strand RNA viruses also interact with host proteins and regulate virus RNA synthesis. Cyclophilin A binds to the N protein and is a required factor for replication of VSVNJ
but not VSVI
). Recently, peroxiredoxin 1 was identified as a binding partner of the nucleocapsid protein of measles virus and was found to be required for its replication and transcription (63
). Hantavirus nucleocapsid protein binds to a ribosomal protein, RPS19, and mediates preferential translation of viral mRNA (14
). Thus, several viral replication proteins appear to interact with cellular proteins to modulate a variety of functions required for successful replication of the virus in the host.
In an attempt to identify the cellular proteins that interact with VSV P protein and understand the functional importance of these interactions, we employed an IP assay to pull down host factors interacting with the VSV P protein and subsequently used MS analysis to identify these proteins. Among the many cellular proteins identified, we focused our studies on PCBP2 and PCBP1, two members of a family of hnRNPs, which are known to regulate transcription, translation, and mRNA stability in cells (46
). In this study, we demonstrated that PCBP2 and PCBP1 function as negative regulators of VSV infection in human cell types, including HeLa and HEK293. Further examination revealed that these two proteins specifically inhibit viral gene expression.
Although PCBP2 and PCBP1 are two isoforms of the same family of proteins with nearly 90% sequence identity (58
), the inhibitory effects of PCBP2 on VSV growth and viral gene expression were significantly more pronounced than those of PCBP1 ( and ). The reasons for this are unknown at this time but could be in part due to the fact that the amount of PCBP1 is lower in the cytoplasm, where VSV replicates exclusively. It is also possible that the inherent differences in their inhibitory activities on VSV could be due to their associations with other cellular interacting partners. Members of the PCBP family are known to form functional multimers with other cellular proteins for their shuttling between the nucleus and the cytoplasm as well as for their functions. PCBP2 is known to interact with at least hnRNP K, I, and L (37
). Furthermore, PCBP2 interacts with myotonic dystrophy type 2 protein ZNF9 in regulating ornithine decarboxylase expression (30
) and associates with the nucleo-cytoplasmic SR protein (SRp20) to modulate poliovirus genome translation (5
), whereas PCBP1 acts together with hnRNP A2 in negatively controlling the expression of mRNA containing A2 response elements (38
). Whether the associations of different cellular proteins with PCBP2 and PCBP1 are responsible for the observed differences in inhibition of VSV growth and gene expression is unknown, and further studies along these lines may help us understand more about the inhibitory effects of these two proteins on VSV. Additionally, PCBP1 and PCBP2 form functional homo- and heterodimers in supporting translation and replication of poliovirus genome RNA. It has been shown that PCBP1 can rescue poliovirus replication when PCBP2 is depleted in the cell (29
), demonstrating a functional cooperation between these two proteins in poliovirus replication. Such cooperation does not seem to exist in inhibiting or enhancing VSV replication, as overexpression or depletion of both proteins simultaneously did not inhibit or enhance VSV growth significantly (data not shown) compared to the individual proteins.
To understand how PCBP2 is involved in inhibiting VSV growth and viral gene expression, we examined whether PCBP2 interfered with the formation of viral replication protein complexes, which are required for transcription and replication of the VSV genome. However, our studies showed that the interaction of PCBP2 with VSV P protein did not interfere with the formation of complexes of N-P or L-P or the homo-oligomerization of P-P protein. A recent study showed that PCBP2 interacts with MAVS and mediates its degradation through the proteasome pathway via ubiquitination by the E3 ubiquitin ligase AIP4 (67
). Since the proteasome pathway is also involved in inhibition of VSV (33
), we examined whether degradation of P through the proteasome pathway by ubiquitination might account for the inhibition of VSV by PCBP2. Although the studies reported here successfully demonstrated for the first time ubiquitination of the VSV P protein and its degradation through the proteasome pathway, the results also sugges that PCBP2 does not directly target the P protein for degradation. So, the inhibition mediated by PCBP2 is independent of the proteasome pathway. Therefore, the exact mechanism by which PCBP2 exerts its inhibitory effect remains to be elucidated. Further studies on PCBP2-interacting proteins may reveal how PCBP2 inhibits VSV growth and gene expression.
The effects of PCBP2 and PCBP1 on replication of other viral pathogens have been found to be variable. For example, PCBP2 binds the 5′-untranslated region (UTR) and enhances translation of the internal ribosome entry site (IRES) containing mRNA of several positive-strand RNA viruses, such as poliovirus, enterovirus, hepatitis A virus, and coxsakievirus (24
). On the other hand, its binding to the 5′-UTR of HCV has no effect on translation of HCV IRES-containing genomic RNA (28
). Additionally, PCBP1 but not PCBP2 mediated a strong inhibition of gene expression steps in HIV-1 infection (66
). While a role for PCBP2 and PCBP1 has not been reported for replication of any of the negative-strand RNA viruses yet, our findings that PCBP2 and PCBP1 are antagonists for VSV infection provide the first example of the involvement of these proteins in gene expression of this class of RNA viruses. The results underscore the complexity of PCBP regulatory functions in virus-infected cells. PCBP2 is induced following virus-mediated IFN induction. Although it downregulates the IFN response stimulated by viral pathogens (67
), in this study we showed that it suppresses VSV growth by targeting viral gene expression. Other interferon-induced proteins, such as IFITM3, tetherin, and Ars2, have been shown recently to inhibit VSV replication at different stages of infection. While Ars2 (54
) restricts VSV replication at the level of viral RNA synthesis, IFITM3 targets an early event after endocytosis and before primary transcription, and tetherin blocks virion budding from infected cells (64
). Thus, the host cells employ multiple and redundant mechanisms to restrict replication of an invading pathogen. In concordance with a previous report (67
), we also observed an increase of PCBP2 protein level in VSV-infected cells (data not shown). Thus, PCBP2 seems to restrict VSV replication at the level of primary and secondary transcription as well as genome replication, whereas the role of PCBP1 in VSV replication appears to be more restricted. While the viral genome replication was not adversely affected by PCBP1, only the primary transcription was affected negatively by this protein. Additional studies will be needed to understand further the role of PCBP1 in inhibiting VSV primary transcription. Moreover, oligoadenylate synthetase (OAS) and RNase L, which are also induced by IFN (57
), are involved in blocking VSV protein synthesis and thus virus replication (1
). Since PCBP2 mRNA is the activator for OAS (44
), it would be interesting to examine the relationship between PCBP2, OAS, and RNase L in restricting replication of VSV and other viruses.
The findings reported here add to the ever-increasing list of functions reported for PCBP2 and PCBP1. We have demonstrated the effects of PCBP2 and PCBP1 at the level of viral gene expression during VSV infection. It is possible that PCBP2 and PCBP1 affect the viral mRNA transcription process itself, or mRNA stability, or both. PCBP2 and PCBP1 have been implicated in negatively regulating the stability of p21 mRNA (61
). Furthermore, both PCBP1 and PCBP2 have been shown to be components of stress granules (SG) and processing bodies (P-bodies), and thus they have been proposed to participate in targeting newly synthesized mRNA to P-bodies for storage and/or degradation (26
). It has been reported that siRNA-mediated depletion of TIA-1, a component of SG, results in increased replication of VSV (40
). Our preliminary data suggest that VSV induces SG formation in infected cells (P.X.D. and A.K.P., unpublished data). It is possible that PCBP2 and PCBP1 directly interact with viral RNA species (like the genome, antigenome, mRNA, and leader and trailer RNAs) to sequester them in SG, thereby inhibiting viral gene expression, or that their binding impedes the accessibility of the P-L complex to viral RNAs and thus reduces viral gene expression.
In summary, our results demonstrate the antagonistic effects of PCBP2 and PCBP1 on VSV infection in cell culture systems. The inhibitory effects were shown to be at the level of viral gene expression. The molecular mechanism(s) by which PCBP1 and PCBP2 exert their inhibitory effects on VSV RNA synthesis are under investigation.