Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

York, Joanne; Nunberg, Jack H.

doi:10.1128/jvi.01124-16

Cited by 27 publications

(25 citation statements)

References 69 publications

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“…Interestingly, we found that A168 had reverted to the wild-type T168 in 55% of reads. This back-mutation was previously noted upon serial passage of the ΔG2-GPC rCan mutant in standard cell culture medium (28), suggesting that the presence of the N166 glycan enhances virus fitness in cell culture. The V353A substitution in GP2 (39% of reads) is also noteworthy due to its proximity to the I347F change noted upon passage of K33S-GPC rCan in the absence of AmCl.…”

Section: K33s-gpc Rcan Retains K33s and F427i Upon Prolonged Passage supporting

confidence: 62%

“…After 10 passages, the p10 virus was subjected to RNA-Seq. Other than the previously noted A-to-T transversion in the S RNA 3=-UTR (28), no additional nucleotide changes were identified ( Table 2). The stability of rCan perhaps is not surprising because Candid#1 had been extensively passaged in Vero cells.…”

Section: K33s-gpc Rcan Retains K33s and F427i Upon Prolonged Passage mentioning

confidence: 65%

“…In order to investigate this point further, we examined the sensitivity of the Candid#1 virus to pH-induced inactivation. We employed reverse genetics (27,28) to rescue a recombinant Candid#1 virus (rCan) and subjected the virus to low pH to trigger the abortive fusogenic reorganization in GPC. Exposure to pH 5.0 abrogated the…”

Section: Resultsmentioning

confidence: 99%

“…As F427 renders certain K33 mutants unable to mediate membrane fusion at endosomal pH, we surmised that reversion would be lethal in a Candid#1 virus bearing the appropriate K33 mutation. To test this notion, we engineered the K33S substitution into rCan using reverse genetics (27,28). This mutation was chosen due to the severity of the fusion defect in the presence of F427 (20) and to take advantage of the fact that two nucleotide changes would be required for back-mutation to wild-type K33.…”

Section: Figmentioning

confidence: 99%

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

York

Nunberg

2018

J Virol

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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.

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Section: K33s-gpc Rcan Retains K33s and F427i Upon Prolonged Passage supporting

confidence: 62%

Section: K33s-gpc Rcan Retains K33s and F427i Upon Prolonged Passage mentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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

York

Nunberg

2018

J Virol

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“…First, the N-terminal myristoylation of SSP, although not critical for assembly, trafficking, nor localization with specific lipid domains of the cell surface, [141,185] is critical for fusion with host cell membranes [191]. Second, the penultimate C-terminal C57 is a critical contributor of a zinc-finger motif in the cytoplasmic tail of GP2, which is required to secure the SSP with the GPC [186,192,193].…”

Section: The Stable Signal Peptide -Sspmentioning

confidence: 99%

Arenaviruses

Brouillette¹

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To my husband, Marc, for his endless encouragement and support iii ACKNOWELDGEMENTS First, I would like to thank my mentor, Dr. Wendy Maury. Without her, none of this would have been possible. I would also like to thank all the people in the Department of Microbiology with whom I have worked over the last four years and my committee members who took the time to meet with me about my work each year.Particularly, I would like to acknowledge Nick Lennemann, Beth Rhein, and Sven Moller-Tank. Sven was always a source of amusement and good advice. Nick taught me essentially everything I needed to know to succeed and helped me start my first project.Lastly, Beth Rhein is a great friend. I cannot imagine my graduate career without her. I would also like to make special note of an undergraduate research assistant who worked tirelessly by my side, Elisabeth Phillips. She regularly exceeded her responsibilities and assisted me with countless experiments needed for my dissertation.Her dedication and work ethic are unmatched.Finally, I would like to thank my friends and family for their love and support. In particular, I thank my husband and best friend Marc. He has always been indispensable. iv ABSTRACTThe family Arenaviridae consists of over 30 members, some of which can infect humans and cause severe hemorrhagic fever. The three arenaviruses studied in this thesis, Machupo virus (MACV), Junín virus (JUNV), and Lassa virus (LASV), are causative agents of Bolivian hemorrhagic fever, Argentine hemorrhagic fever, and Lassa fever, respectively. Epidemics of these diseases can carry high rates of morbidity and mortality, and due to a lack of available countermeasures, all three viruses are considered category A priority pathogens by the CDC.Arenavirus glycoproteins (GPCs) are considered class I viral fusion proteins, but in multiple regards, they are quite unusual for viral envelope proteins. The GP precursor is translated as a polypeptide that is proteolytically processed within the secretory pathway by two sequential cleavage events to produce a tripartite complex (heterotrimers) that assemble into homotrimers to form heterononamers. The three distinct units of the GPC structure are a receptor binding domain, GP1, and fusion domain, GP2, and most peculiarly, a stable signal peptide (SSP) that traverses the membrane twice and associates with the GP2.The glycoprotein complex (GPC) of MACV encodes nine putative sites for Nlinked glycosylation. As N-glycans have been shown to be important for a multitude of factors in glycoprotein biology, we sought to investigate the potential roles of the Nglycans on MACV GPC expression, function, antigenicity, and immunogenicity. To do so, we used MACV GPC-VSVG-eGFP pseudovirions (MACV-VSV) to study the effects of both individual and combinatorial N-glycan losses. Our results demonstrate that loss of N-glycans at sites N178, N370, or N378 resulted in a loss of GPC proteolytic processing, while the accumulation of multiple N-glycans reduced total GPC expression and function. Replacement of the ...

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Characterization and epitope mapping of a panel of monoclonal antibodies against HIV‐1 matrix protein

Zhang

Feng

Bai

et al. 2018

Biotech and App Biochem

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The HIV-1 Gag precursor protein (p55) is the main structural protein comprising the matrix (MA/p17), capsid (CA/p24), and nucleocapsid (NC/p7) proteins, and is uniquely responsible for virion assembly within the virus life cycle. The MA protein plays a critical role in plasma membrane targeting and envelope glycoprotein (Env) uptake during virion assembly. Yet, when viral infection occurs, the MA protein may also be involved in virion uncoating, dissociating from the plasma membrane, and participating in the nuclear importation process. Thus, the MA protein contains a reversibly membrane-binding signal and varied conformation to govern its subcellular localization and biological functions. However, these purported different conformations of the MA protein during assembly are poorly understood, especially in terms of its function as a component of the precursor protein. In this study, we characterized a panel of monoclonal antibodies against MA that showed discrete reactivity to p55, an intermediate (p41), and the final p17 mature form. We suggest that these antibodies could be used to track the different conformations of MA during the HIV-1 life cycle, particularly during HIV-1 assembly and maturation, and contribute to structure determination of MA or MA precursors. These antibodies would also have clinical value, including serving for therapeutic strategy to interfere AIDS progression, reagent in diagnostic kit for the detection of virion-free p17 or p17 derived from virion lysate.

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Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

Cited by 27 publications

References 69 publications

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

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

Arenaviruses

Characterization and epitope mapping of a panel of monoclonal antibodies against HIV‐1 matrix protein

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