Epistastic Interactions within the Junín Virus Envelope Glycoprotein Complex Provide an Evolutionary Barrier to Reversion in the Live-Attenuated Candid#1 Vaccine

Abstract

The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here, we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers.

A potent Lassa virus antiviral targets an arenavirus virulence determinant

“…Selection for mutations that destabilize the GP complex in the presence of LHF-535 is consistent with the proposed mechanism of action, which is stabilization of the GP complex and inhibition of the conformational rearrangement required for fusion [15]. Also supporting this mechanism of action is a recent report of an epistatic interaction between positions K33 of the Junín virus stable signal peptide (SSP) and F427 within GP2 [35]. Although K33 is highly conserved across mammalian arenaviruses, engineered mutations at this position modulate sensitivity to entry inhibitors [14], further implicating interactions between SSP and GP2 in the antiviral mechanism.…”

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

“…Later, 100% of the MCg1-infected mice contained a population of GPC A168S/T after 42 days of infection ( S2 Table ). Mutations were not observed after five serial passages of MCg1 in vitro [ 37 ] and did not affect MCg1 growth in vitro , which is important for development of a genetically stable and attenuated vaccine candidates [ 30 ]. No mutations occurred in MCg1 in cell culture experiments in our study.…”

“…An effective vaccine against JUNV, the Candid#1 (Cd#1) live attenuated vaccine, is available in Argentina and has substantially reduced AHF cases within endemic areas [ 28 , 29 ]. The Cd#1 strain was developed by passaging the pathogenic JUNV XJ strain twice in guinea pigs and 44 times in suckling mice brains [ 30 , 31 ]. The resulting strain (XJ44) was attenuated in guinea pigs but still virulent in suckling mice.…”

Biological Characterization of Conserved Residues within the Cytoplasmic Tail of the Pichinde Arenaviral Glycoprotein Subunit 2 (GP2)

“…Since the arenaviral SSP has been shown to be necessary for the intracellular trafficking of GP through the ER and Golgi apparatus for its proper processing and surface expression (22,30,38,39), amino acid substitution mutations at the zinc-binding residues of the GP2 CT may disrupt its association with the SSP and thus can impair GP processing and membrane fusogenic activity (26). A functional interaction between the SSP and GP2 has also been supported by earlier studies demonstrating that the SSP and GP2 transmembrane and CT domains from the same virus are needed for proper GP maturation and production of infectious viruses (31) and that an epistatic interaction between the attenuated mutation F427I in GP2 and the SSP K33S mutation of JUNV Candid#1 is required for regulating membrane fusion activity (40). Taking these data together, most of the conserved residues of the GP2 CT are probably required for its proper folding and interaction with the SSP, which facilitates GP1-GP2 cleavage, intracellular trafficking, cell surface expression, and fusion activity.…”