Because of their limited genome coding capacity, viruses depend on host factors to carry out important functions. During the course of evolution, each virus has acquired a unique array of multifunctional proteins. The arterivirus nsp1 turns out to be one such protein. Previous studies on nsp1 of EAV and PRRSV have suggested that this protein is a major regulator of virus replication as well as a potent suppressor of host innate immune response (3
). In this article, by using PRRSV nsp1β as bait, we identified several cellular proteins that interact with this viral protein, and these cellular proteins may play an important role(s) in the viral life cycle. Although the studies reported here focus on PCBPs, our IP-MS results have identified several other proteins of interest (). Specifically, several hnRNP proteins, e.g., hnRNP K and hnRNP A2/B1, were identified, which have been shown to be important for replication of other viruses such as coronaviruses (25
). Another protein of interest is the cellular poly(A) binding protein cytoplasmic 4 (PABP4), which is known to be expressed upon T-cell activation and is involved in RNA stabilization and translation (32
). A common property among these putative nsp1β-interacting proteins is their inherent RNA binding ability. It will be interesting to explore the possible involvement of these cellular proteins in PRRSV life cycle, particularly in viral genome replication and transcription. As nsp1β has also been predicted to interact with RNA, these proteins together with nsp1β might form RNP complexes and play important regulatory roles in virus life cycle (30
The nsp1β-PCBP interaction was confirmed both under ectopic expression conditions and in PRRSV-infected cells. Although RNase treatment did not alter nsp1β-PCBP1/2 interaction, it is possible that these interacting partners might interact through a specific RNA moiety that is protected from RNases. The observation of partial colocalization of PCBP1/2 to viral RTC upon PRRSV infection of cells is consistent with a recent report, which suggests that only 25% of total EAV nsp1 in infected cells is present in the cytoplasmic RTC-containing cellular fraction (50
). Together with nsp1β's presumed role in sg mRNA synthesis, these results indicate possible recruitment of PCBPs to the viral RTCs through interactions with PRRSV nsp1β for viral RNA synthesis. Additionally, nsp1β and PCBP1/2 were also found to colocalize in the nucleus (). Both nsp1α and nsp1β of PRRSV are known to translocate to the nucleus during late times of infection (7
). The functional significance of the interaction of these proteins in the nucleus is unclear, but it is possible that these complexes might regulate cellular RNA metabolism in the nucleus.
Two arteriviral proteins, nsp2 and nsp3, are implicated in modifying the endoplasmic reticulum (ER) membrane to form double membrane vesicles (DMVs), which are the sites for viral genome amplification (40
). Since PRRSV nsp2/3 and the cytoplasmic fraction of PCBP1/2 localize together in virus-infected cells (), we attempted to verify this interaction by co-IP experiments. Such experiments did not yield unequivocal results, indicating that the association might be a indirect, weak, or transient one that is not readily detectable by routine co-IP procedures. On the other hand, the interaction of the PRRSV nsp9 (the viral RdRp) with PCBP1/2 could be readily demonstrated when these proteins were expressed ectopically. Demonstration of a similar interaction in virus-infected cells will be important for understanding the functional consequences of this interaction in viral genome replication.
Arteriviral 5′UTR contains cis
-acting sequences required for genome replication and sg mRNA production (49
). The PCBPs have been reported to bind both 5′UTR and 3′UTR of cellular as well as viral genes and affect RNA metabolism. Binding of PCBP1 and PCBP2 to the promoter of mouse mu opioid receptor and BRCA1 enhances their transcription (21
). PCBP2 has been shown to bind the 3′UTR of tyrosine hydroxylase mRNA and increase mRNA stabilization and translation (56
). A previous study had identified four MA104 cell proteins, with approximate molecular masses of 103, 86, 55, and 36 kDa, of unknown identity interacting with SHFV 3′UTR of (−)-sense genome (which is antisense to viral 5′UTR) (16
). Using gel mobility shift assays, we have also demonstrated that PCBP1 and PCBP2 bind to PRRSV (+) 5′UTR. As the molecular masses of PCBPs are ~40 kDa, it is possible that one of those proteins bound to SHFV (−) 3′UTR might be PCBP. The 191-nt 5′UTR of type II PRRSV contains several pyrimidine-rich stretches, which are considered possible sites for PCBP binding. Especially, the 3′ terminal 15 nt (from nt 177 to nt 191) of the 5′UTR, which are highly conserved across arteriviruses (49
), have strong homology (only 2 nt difference) with the consensus PCBP binding site on cellular mRNAs (15
). It is possible that PCBPs may bind specifically to these conserved sequences to modulate viral genome transcription. It would be interesting to investigate if the PCBP-nsp1β interaction is required for PCBP's binding to PRRSV 5′UTR or if these are two mutually exclusive events.
Using an siRNA-mediated silencing approach, we have established the importance of PCBPs for PRRSV genome replication/transcription. Both PCBP1 and PCBP2 are closely related (~90% amino acid identity). Although these two proteins are known to have overlapping functions (52
), our results presented here indicate that they might be involved in yet-unknown nonoverlapping activities in PRRSV replication, as the simultaneous depletion of both proteins led to an additive reduction in viral growth compared to individual depletion (C). PCBP1 and PCBP2 do not appear to be involved in translation from genomic RNA. However, genomic and antigenomic RNA synthesis was reduced in the PCBP1/2-deficient cells (A). The observation that PCBPs bind specifically to the 5′UTR of PRRSV prompts a critical question related to viral RNA synthesis: how does this binding influence genome replication, which initiates from the 3′UTR? Studies from poliovirus replicative cycle may offer an interesting explanation in this regard. Binding of PCBP1 and PCBP2 to the 5′ cloverleaf (5′CL) structure of poliovirus stimulates the association of viral polymerase precursor (3CDpro
) with the 5′CL (13
). Binding of poly(A) binding protein (PABP) to the 3′UTR poly(A) tail and its interaction with the PCBP2 and 3CDpro
then lead to genome circularization and replication initiation (14
). Such genome circularization has been suggested as a universal mechanism for (+)-strand RNA virus replication, including that of coronaviruses (41
). It is possible that the PCBPs might be an integral part of such an RNP complex that is required for the initiation of negative-strand RNA synthesis in PRRSV.
The inhibitory effect on sg RNA synthesis upon PCBP1/2 depletion could be the result of reduced levels of full-length genome and antigenome synthesis or could be due to a loss of direct interaction of PCBP1/2 with transcription-controlling cis-acting elements within the viral genome. Nidoviral sg RNAs are produced through the process of discontinuous transcription, which has been proposed to involve physical interaction between the leader transcription-regulating sequence (TRS-L) and the body TRSs (TRS-B), which are present upstream of each ORF within the viral genome. It is possible that PCBPs might interact with TRS-L and TRS-B and might help to bring together these distant RNA moieties. Further studies will be required to address the interactions of PCBP1/2 with the TRSs and how these interactions might regulate sg RNA synthesis in PRRSV-infected cells.
Overall, our studies reveal two cellular factors, PCBP1 and PCBP2, as being important regulators of PRRSV replication. They both have affinity for viral 5′UTR and interact with two viral replicase proteins. Whether PRRSV's dependence on PCBPs is also true for other arteriviruses needs further investigation. Considering the similarity in their replication strategies, these cellular proteins might also be important for other members of the Nidovirales order.