In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation

Abstract

Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membranespanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly. IMPORTANCEResearchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site. Formation of infectious retroviral particles requires a symphony of viral and host cell proteins to coordinate in a spatial and temporal manner. Typically, this is an exclusive process wherein components from a given virus assemble only with components from the same or closely related viruses, such as HIV-1 and HIV-2 (1-5). However, this exclusivity is not typical for the incorporation of viral glycoproteins. Incorporation of foreign glycoproteins into nonnative viral particles is termed pseudotyping (6-8), and researchers from numerous fields routinely exploit this ability to manipulate viral tropism. Although very little is known about the molecular mechanisms that govern this process, it is clearly not random. For example, we have shown using scanning electron microscopy (SEM) that both vesicular...

FASTAptameR 2.0: A Web Tool for Combinatorial Sequence Selections

“…The Distance module is a new feature of FASTAptameR 2.0 that computes the Levenshtein edit distance (LED) between a query sequence and every other sequence in the population. An in-house precursor of this module was previously used in an in vivo selection [60]. Distance analysis can be especially useful when monitoring the accumulation of point mutants, evaluating the effectiveness of a mutagenesis protocol, or monitoring diversity near the beginning of a selection from sequences that densely sample local sequence space ( e.g., via mutagenic PCR or doped resynthesis).…”

HIV-1 Vif Triggers Cell Cycle Arrest by Degrading Cellular PPP2R5 Phospho-regulators

“…Library synthesis was modified from previous methods (Salamango et al, 2016) and is depicted in Figure S1. Mutagenic oligonucleotide primers were generated by IDT with a mutation frequency of 3% per nucleotide position.…”

Deep Mutational Scanning of Viral Glycoproteins and Their Host Receptors

“…The general approach of tracking replication of virus variants through deep sequencing has been used to extensively characterize the glycoproteins of two viruses highly relevant to public health: HIV-1 (Haddox et al, 2016(Haddox et al, , 2018Dingens et al, 2017Dingens et al, , 2019a and influenza A (Thyagarajan and Bloom, 2014;Wu et al, 2014Wu et al, , 2017aWu et al, , 2020Doud and Bloom, 2016;Canale et al, 2018;Lee et al, 2018). There have also been many efforts within the past few years to apply this selection strategy and others more broadly to other viruses, including murine leukemia virus (MLV) (Salamango et al, 2016), Zika virus (ZIKV) (Gong et al, 2018;Setoh et al, 2019;Sourisseau et al, 2019), and most recently, SARS-CoV-2 (Greaney et al, 2020;Linsky et al, 2020;Starr et al, 2020;Chan et al, 2021). Below, we introduce and discuss examples of selection strategies used for deep mutagenesis of viral glycoproteins.…”

“…Multiple selection strategies can be implemented for the same deep mutagenesis library to determine the effects of mutations separately on different properties of a viral fusion protein or glycoprotein. Salamango et al (2016) utilized three different selection strategies on deep mutational scans of MLV Env for infectivity, Env fusion activity, and incorporation into viral particles. The mutational landscapes for the strategies were compared to filter out how specific sites and their mutations influence each selected property.…”

“…Mutations in MLV Env were discovered with defective infectivity because of poor incorporation, despite the Env mutants remaining active for membrane fusion. These MLV Env mutants were poorly incorporated into pseudotyped HIV-1 particles, possibly due to changes in lipid interactions at assembly sites (Salamango et al, 2016). Pseudotyping allows for the manipulation of cell type tropism through the incorporation of envelope glycoproteins from one virus into alternative virus backgrounds.…”