For many negative-strand RNA viruses, it has been shown that self-association of the virus nucleoprotein (NP) is required for the formation of the ribonucleoprotein core that directs both viral RNA replication and gene transcription (1
). In this work, we have demonstrated that the NP of the prototypic arenavirus LCMV, as well as the NPs of OW LASV and NW MACV HF arenaviruses, has the property of self-association (homotypic interaction), a finding consistent with recent reports documenting the ability of the LASV (40
) and NW arenavirus TCRV (23
) NPs to form oligomeric structures. In addition, we have also documented heterotypic interactions among NPs from different arenavirus species.
The robustness of LCMV NP homotypic interaction was supported by obtaining similar findings using Co-IP () and M2H () assays. In addition, homotypic LCMV NP interaction was also confirmed by BiFC () and shown to be restricted to the cell cytoplasm, as predicted based on the replication cycle of arenaviruses (7
). In both the M2H (data not shown) and the BiFC assays, LCMV NP-NP interaction was detected only when the N-terminal domain of NP was untagged, suggesting a potential role of the N terminus in NP self-association. This NP self-association was also shown in NPs of OW LASV and NW MACV HF arenaviruses by Co-IP () and M2H () assays. In addition, we have also documented heterotypic interaction among NPs from different arenavirus species. Results from assays using a collection of LCMV NP N-terminal () and C-terminal () deletion mutants indicate the importance of the N-terminal domain of the LCMV NP for its self-association. Recently published findings for the TCRV NP showed that residues 19 to 119 in the N-terminal domain played a critical role in self-association of the TCRV NP protein. Our findings with the LCMV NP further confirm that residues 1 to 50 at the N terminus, as well as residues located within a region covering amino acids 308 to 358, are critical for LCMV NP self-association.
Our results have also shown that the NP self-association domain does not overlap with a previously identified domain responsible for the anti-IFN function of the LCMV NP (). Accordingly, these two functions can be separated physically in the LCMV NP structure. Thus, LCMV NP mutants lacking the anti-IFN activity interacted with the wild-type LCMV NP with strength similar to that of the wild-type NP-NP interaction. This is further supported by our previous finding that N-terminal deletion mutants affecting NP-NP interaction (e.g., deletions up to the 350 first amino acids) retained their ability to inhibit Sendai virus (SeV)-mediated induction of type I IFN (24
). This, in turn, suggests that monomers of the NP could counteract the cellular host IFN response, raising the intriguing possibility that defined NP fragments often observed in virally infected cells may, despite not contributing to formation of functional RNPs, favor virus propagation by providing a decoy strategy against the host innate immune response (9
Our results and previous reports (23
) suggest the presence of two domains in arenavirus NPs in which the C-terminal domain is responsible for counteracting the host type I IFN response (24
) and also for interaction with Z (23
), with key residues involved in these two functions (37
). On the other hand, the N-terminal domain would be involved in NP self-association (reference 23
and this study). However, a contribution of residues within the C-terminal region of the NP in NP-NP interaction cannot be ruled out. For instance, relevant residues within the C-terminal region of the NP may participate in stabilizing the N-terminal domain involved in the NP-NP interaction or the NP interaction with other viral or cellular proteins that create the appropriate structural conformation for the NP-NP interaction. This could explain some differences found in the magnitude of the interaction among C-terminal deletion mutants as well as differences observed in heterotypic NP-NP interactions. We (and others) have recently demonstrated a similar situation with the influenza IFN antagonist nonstructural protein 1 (NS1) (10
). A second domain (amino acids 103 and 106) in the middle of NS1 was identified for binding to the cleavage and polyadenylation specificity factor (CPSF) in combination with the conventional CPSF interaction site located at the C terminus of NS1 (32
Based on the recently described crystal structure of the LASV NP, Qi et al. suggested that NP self-association occurs through N- to C-terminal interactions (40
). At first glance, our results (and those found with the TCRV NP [23
]) are difficult to reconcile with this model. It is plausible that self-association of arenavirus NPs may require interaction between other interfaces in addition to those suggested by the crystal structure and for which the N-terminal region may play a major role. One potential scenario that would help to reconcile both models is that NP self-association is mediated by two different mechanisms. One involves head-to-tail interactions (as suggested by the crystal structure model) and the other involves an RNA-based bridge through the N-terminal domain of the NP (as suggested by the biochemical results). This model would explain why N-terminal mutants are not able to self-associate or interact with RNA. On the other hand, in this model, C-terminal mutants would lose the NP-NP interaction domain at the C-terminal end but still be able to interact with RNA and, therefore, still be able to show NP self-association. Recently, the LASV NP quaternary structure has been determined (6
). These studies uncovered asymmetric and symmetric trimeric structures of the LASV NP with a tail-to-tail (N-N) interface and a head-to-tail (C-N) interface, respectively. Electron microscopy and small-angle X-ray scattering analysis favor the NP association into a symmetric complex in solution, suggesting that C-N interactions reflect true interactions between NP monomers in solution without excluding the possibility that upon binding to RNA or after posttranslational modifications, the NP could reorganize, forming other structures (6
). Future studies are required to demonstrate this hypothesis and to explain these apparent discrepancies between biochemical results and predictions derived from the crystal structure of the LASV NP. Although in our assays we did not include a specific LCMV RNA sequence that can facilitate NP self-association, we cannot exclude the possibility that LCMV NP mRNA produced by the pCAGGs expression plasmid is specifically recognized by the LCMV NP. It should be noted that this nucleus-generated, plasmid-derived LCMV NP mRNA contains features, including a distinct 3′ untranslated region (UTR) and poly(A) tail, that are missing in the viral mRNAs produced in the cytoplasm of LCMV-infected cells. Alternatively, the interaction of the NP with RNA may not require viral RNA and could be mediated by an unspecific cellular RNA. Whether NP self-association is required for binding RNA and whether monomers of NP bind to RNA remain to be determined. Intriguingly, previous studies with the TCRV NP suggested that NP-NP interaction was RNA independent, a result which appears counterintuitive for a negative-strand RNA virus.
We have previously shown that N-terminal deletions in the NP abrogated its ability to promote RNA replication and gene expression of an LCMV minigenome (MG) (24
). This finding may, at least partly, reflect an inability of these NP mutants to generate functional nucleocapsids due to their lacking the self-association property. It needs to be further evaluated whether NP self-association is required for RNA binding and transcriptional activities of RNP complexes. Our results also showed that deletions in the C-terminal region of the NP did not affect NP self-association, but C-terminal deletions were shown to abrogate NP functions required for replication and transcription of an LCMV minigenome (24
). These results suggest that the C-terminal region of the NP might have a direct role in transcription and replication that is independent of nucleocapsid formation. As previously reported (24
), the overall structural integrity of the LCMV NP is probably required for its involvement in replication and transcription.
NPs are highly conserved among members in the Arenaviridae
family, and comparisons between the LCMV NP and the LASV NP and between the LCMV NP and the MACV NP show 61% and 50% amino acid sequence identities, respectively, that increase to 66% and 56% when comparing only the 350-amino-acid N-terminal region (ClustalW2 analysis). Our studies have also shown that the LCMV NP interacts with the LASV NP and with the MACV NP, but homotypic interactions were slightly stronger than heterotypic NP-NP interactions, indicating that specific amino acid residues may be required for a proper NP-NP interaction. Our findings, however, suggest a possible protein domain that is common among even distantly related arenaviruses and which may potentially provide a target for antiviral development against all members in the family. The identification of specific amino acid residues within the NP that are directly involved in NP-NP interaction may facilitate the design of peptides able to disrupt NP-NP interaction and affect virus RNA synthesis, as previously documented for other viruses (47
). Likewise, it would be feasible to develop assays amenable to high-throughput screening (HTS) technologies to identify small molecule inhibitors affecting NP-NP interaction that may be active against most, or all, human pathogenic arenaviruses.