In Vitro and In Vivo Characterizations of Pichinde Viral Nucleoprotein Exoribonuclease Functions

Abstract

Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFNdeficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCEArenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

A Novel Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and Cellular Immunity after a Booster Dose

“…Compared to inactivated vaccines, live viral vaccines are known to induce strong cellular immunity, which is important for combating various human diseases ranging from microbial infections to cancers. Each of the currently available live viral vectors has its pros and cons, and none has proven to be universally suitable for every pathogen (13,34). An ideal viral vector must be safe, induce strong and durable cellular and humoral immune responses, have no preexisting immunity, and, ideally, not lose potency upon repeated application.…”

“…The glycoprotein (GPC), encoded on the small (S) segment, is posttranslationally processed into a stable signal peptide (SSP), the receptor-binding G1 protein, and the transmembrane G2 protein (7). The nucleoprotein (NP) of the S segment encapsidates viral genomic RNAs and is required for viral RNA synthesis and host immune suppression (8)(9)(10)(11)(12)(13).…”

“…We have previously developed a reverse genetics system to generate infectious PICV from two plasmids that produce full-length antigenomic strands of the L and S RNAs (23). Using this reverse genetics system, we have characterized the functional mechanisms of viral virulence determinants and the biological roles of arenaviral proteins (13,(24)(25)(26)(27)(28).…”

Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain

“…Furthermore, even though arenavirus pathogens encode both NP and Z as strong IFN antagonists (11, 12, 14-17, 22, 25), the roles of IFNs in arenavirus-caused diseases remain controversial. LASV infection is associated with a low level of IFN responses (26,27), and type I IFNs have been shown to control LASV, LCMV, and PICV replication (18,(28)(29)(30)(31)(32). However, NW arenaviruses such as MACV and JUNV have been shown to induce high levels of IFNs in A549 cells and human DCs (33, 34) but not in human macrophages and monocytes (10).…”

“…Several studies have shown that LASV and JUNV, but not their nonpathogenic counterparts Mopeia virus (MOPV) and Tacaribe virus (TCRV), can inhibit human macrophage activation (7)(8)(9)(10), the molecular mechanism of which is unknown. As all arenaviruses encode a conserved nucleoprotein (NP) RNase with activity to block the interferon (IFN) induction (11)(12)(13)(14)(15)(16)(17)(18), it seems unlikely that NP mediates the differential inhibition of macrophages by various arenaviruses. We have recently found that the Z proteins from known arenavirus pathogens, including HF-causing LASV and JUNV as well as lymphocytic choriomeningitis virus (LCMV), that can cause neurologic diseases (19), but not those from others, such as TCRV and Pichinde virus (PICV), which have not been associated with significant human diseases (20,21), can inhibit RIG-i/MDA5 and block the RIG-i-like-receptor (RLR)-dependent IFN production in macrophages and that the differential RLR inhibition is determined by the N-terminal domain (NTD) (22).…”