family members, only a few can cause virulent infections in unnatural hosts, such as arenavirus-induced hemorrhagic fevers in humans, the reasons of which are largely unknown. A postulated hypothesis proposes that the viral genome may encode critical determinants for virulence. We use a safe small animal model, in which two PICV strains derived from low (P2) and high (P18) passages in guinea pigs can respectively cause avirulent and virulent infections with opposing outcomes in the animals, to characterize the virulence-associated determinants in arenavirus genome. Compared to the variable mortality rates of outbred guinea pigs infected with virulent PICV in previous studies (Jahrling et al., 1981
; Scott and Aronson, 2008
), we have observed more consistent high mortality rate and hemorrhagic fever symptoms in our infected animals (–), which is likely due to different experimental settings between our study and others. Knowledge of virulence mechanisms learned from the PICV-guinea pig model can be extrapolated to other pathogenic arenaviruses in humans. Through site-directed mutagenesis and comprehensive characterization of the mutant viruses in this study, we have shown that the E140 residue within the receptor-binding GP1 subunit is an essential virulence determinant of P18 infection of guinea pigs, as the E140K substitution can significantly reduced viral replication and pathogenesis in vivo (–). We have also provided evidence to suggest that the molecular mechanism by which the E140 residue enhances viral replication and virulence is possibly due to its ability to enhance cell entry efficiency as demonstrated in the pseudotyped lentiviral system ().
Using pseudotyped VLPs, we have shown that GPC of virulent P18 virus can mediate a much more efficient cell entry than that of avirulent P2 into a variety of cell lines (data not shown). The correlation of cell entry efficiency and viral virulence in vivo have led us to believe that GPC-mediated cell entry may be an important virulence mechanism of arenavirus infection in vivo. As P2 and P18 GPC proteins differ at three amino acids, 119, 140, and 164, within the G1 subunit that mediates specific binding to host receptor(s) (Lan et al., 2008
), we hypothesize that these residues can affect virus-cell binding to influence the virus entry efficiency. By systematic mutagenesis, we learn that the K140E substitution with a change from positive to negative side chain of the amino acid during the P2→P18 adaptation is essential in enhancing cell entry, which can be further augmented by the S119T or I164V substitution (). However, the exact molecular mechanism by which these amino acid substitutions enhance GPC-mediated cell entry requires a better understanding of PICV GPC interaction with its host receptor(s) at the atomic level, information of which is not available at the moment. The entry receptor of PICV as well as other members of Clade A NW arenaviruses has not yet been identified, although we have produced preliminary data to suggest that PICV does not primarily use a-DG or TfR1 for entry (unpublished data).
Additionally, we have provided evidence to demonstrate the important role of the E140 residue in mediating PICV replication and virulence in vivo. We have shown that a single amino acid substitution of E140 with the P2-specific residue K in the genome of virulent rP18 virus has improved survival rate of infected animals (). Furthermore, this E140K single substitution has led to significantly lower level of viral replication in vivo by at least 2 logs in blood and by 1–4 logs in various organs (), and subsequently less liver pathogenesis (), even in the moribund animals. Our in vivo data have provided evidence for E140 as one of the important virulence determinants of P18 infection in guinea pigs, which correlates well with its role in increasing GPC-dependent virus entry in vitro. It is noteworthy that compared to the pronounced 1–4 log difference in viral replication in vivo (), the GPC mutant viruses replicate only slightly less than the parental rP18 virus by < 0.5 log in vitro (). This could be explained by the differences between the in vivo and in vitro experimental conditions, among which host immunity may play an important role in amplifying the differential infectivity of viral strains in vivo. As early target cells of arenavirus infections are macrophages and DCs, which are immune cells important for both innate and adaptive immune responses, differential entry efficiency into these early target cells due to GPC mutations not only can lead to intrinsically different viral replication levels but may also affect host immunity that in return leads to even greater differences in viral growth in vivo. Regardless of the mechanisms, our data show that the GPC-mediated differential cell entry into host target cells leads to the differences in viral growth in vivo.
This GPC-dependent virulence mechanism is also supported by a recent report demonstrating that a single mutation in LCMV GPC is necessary and sufficient for receptor binding, dendritic cell infection, and long-term persistence (Sullivan et al., 2011
). It is important to note, however, that mutations in GPC alone did not reduce PICV viral virulence to the level of rP2 (–), suggesting the contribution of other possible viral virulence factors besides GPC. We have recently produced evidence to demonstrate that both viral L polymerase and nucleoprotein (NP) may also play important roles in mediating virus virulence ((Liang et al., 2009
) and our unpublished data). Efforts to characterize the virulence-associated mutations in the NP and L proteins are currently underway. Taken together, our study suggests that virulent arenavirus infections are a complex process that requires the synergetic functions of different viral proteins, including but not necessarily limited to GPC, to increase viral entry and RNA synthesis, and to effectively evade host immune detection (Fan et al., 2010
; Martinez-Sobrido et al., 2006
; Qi et al., 2010