Evolution of Recombinant Lymphocytic Choriomeningitis Virus/Lassa Virus In Vivo Highlights the Importance of the GPC Cytosolic Tail in Viral Fitness

Abstract

A key characteristic of arenaviruses is their ability to establish persistent infection in their natural host. Different factors like host age, viral dose strain, and route of infection may contribute to the establishment of persistence. However, the molecular mechanisms governing persistence are not fully understood. Here, we describe gain-of-function mutations of lymphocytic choriomeningitis virus (LCMV) expressing Lassa virus (LASV) GP, which can prolong viremia in mice depending on the sequences in the GP-2 cytoplasmic tail. The initial mutant variant (rLCMV/LASV mut GP) carried a point mutation in the cytosolic tail of the LASV glycoprotein GP corresponding to a K461G substitution. Unlike what occurred with the original rLCMV/LASV wild-type (wt) GP, infection of C57BL/6 mice with the mutated recombinant virus led to a detectable viremia of 2 weeks' duration. Further replacement of the entire sequence of the cytosolic tail from LASV to LCMV GP resulted in increased viral titers and delayed clearance of the viruses. Biosynthesis and cell surface localization of LASV wt and mut GPs were comparable. IMPORTANCEStarting from an emerging virus in a wild-type mouse, we engineered a panel of chimeric Lassa/lymphocytic choriomeningitis viruses. Mutants carrying a viral envelope with the cytosolic tail from the closely related mouse-adapted LCMV were able to achieve a productive viral infection lasting up to 27 days in wild-type mice. Biochemical assays showed a comparable biosynthesis and cell surface localization of LASV wt and mut GPs. These recombinant chimeric viruses could allow the study of immune responses and antivirals targeting the LASV GP.T he prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) represents a powerful experimental model used to study the virus-host interaction of noncytopathic viruses and the role of T cells in clearing viral infections in mouse models (1). Infection with several existing strains results in different outcomes causing either transient, acute infection with virus-specific protective immunity or protracted, chronic infection with persisting viremia and immunosuppression (2). LCMV is an enveloped virus comprising two segments (S and L) of ambisense singlestranded RNA. The S RNA encodes the nucleoprotein (NP) and the envelope glycoprotein precursor (GPC) that is posttranslationally cleaved by signal peptidase and the cellular proprotein convertase SKI-1/S1P into the mature virion glycoprotein complex SSP/GP-1/GP-2 (GPs). The L RNA encodes the RNA-dependent RNA polymerase (L) and a small RING finger protein (Z). The GP mediates cell target attachment and fusion. NP and Z cover several functions, including inhibition of the innate immune response and viral particle budding, respectively. NP and L assemble with the two ambisense segments to form the ribonucleoprotein complexes (RNPs), which serve as the templates for transcription and replication. It has been shown that in LCMV the viral polymerase L and GP-1 of the glycoprotein are important determinants for the...

Residues K465 and G467 within the Cytoplasmic Domain of GP2 Play a Critical Role in the Persistence of Lymphocytic Choriomeningitis Virus in Mice

“…In addition, G467 seems to be required for long-term robust virus replication in mice independent of the presence or absence of a functional IFN-I system. These results are consistent with previous findings showing that rCl-13/ LASV-GPC with a single G461 mutation was able to persist in mice for up to 2 weeks, whereas rCl-13/LASV-GPC carrying both K459 and G461 further enhanced viral replication in mice (26). Future studies aimed at phenotypically characterizing rCl-13 with either V465 or K467 mutations will contribute to a better understanding of the roles played by the CD of GP2 in arenavirus persistence.…”

“…1B). Residues V459K and K461G within GP2 played a critical role in conferring on rCl-13/LASV-GPC the ability to persist longer in mice (26). To examine whether the corresponding residues were required for Cl-13 persistence, we generated rCl-13(GPC/VGKS), where K465 and G467 were mutated to V and K, respectively, which corresponded to the residues found in LASV GP2 (Fig.…”

“…In the present work, we have examined the role of residues K465 and G467 within the CD of Cl-13 GP2 in the virus's ability to establish a persistent infection in adult immunocompetent mice. The rationale for these studies stemmed from observations that mutations in the counterpart residues, V459 and K461, of LASV GP2 played a critical role in the early clearance of rCl-13/LASV-GPC in adult immunocompetent B6 mice (26). Our findings have shown that rCl-13 carrying K465V and G467K mutations within the CD of GP2 was unable to persist in mice, which identified these residues as additional viral determinants that play a critical role in the ability of Cl-13 to persist.…”

“…However, a point mutation, K461G, within GP2 that emerged during replication of rCl-13/LASV-GPC in a mouse resulted in a mutant rCl-13/LASV-GPC that persisted for about 2 weeks in adult B6 mice (26). Characterization of additional LCMV/LASV-GPC mutants revealed that the presence of both V459K and K461G mutations within LASV GP2 further increased the virus's ability to replicate in B6 mice (26). These findings led us to investigate whether the counterpart K465 and G467 residues in Cl-13 GP2 played a critical role in Cl-13 persistence.…”

Lymphocytic choriomeningitis virus: ways to establish and maintain non-cytolytic persistent infection

“…LCMV expressing Lassa virus GP could prolong viremia in mice to 2 weeks duration and replacement of entire sequence of Lassa virus cytosolic tail resulted in increased viral titers and delayed clearance of the virus (Sommerstein et al, 2014).…”