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Human immunodeficiency virus (HIV) Gag drives particle assembly. The capsid (CA) domain is critical for Gag oligomerization, and encodes key residues that dictate Gag-Gag interactions and particle morphology. The immature particle morphology of HIV-2 is intriguing different relative to that of HIV-1. To help define the critical determinants for Gag-Gag interactions and investigate the differences between HIV-1 and HIV-2, we have conducted mutagenesis in targeted locations of HIV-2 CA that have been implicated in Gag-Gag interactions. In particular, a panel of 31 site-directed mutants at the HIV-2 CA inter-hexamer interface, intra-hexamer interface and CA inter-domain linker have been created and analyzed for the efficiency of particle production, particle morphology, particle infectivity, Gag subcellular distribution and in vitro protein assembly. Seven conserved residues (L19A, A41, I152, K153, K157, N194, D196) and two non-conserved residues (G38, N127) were found that impact Gag multimerization and particle assembly. Taken together, these observations complement structural analyses of immature HIV-2 particle morphology and Gag lattice organization, and provide insights into the morphological differences between HIV-1 and HIV-2 immature particles and their impact on virus replication.

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Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we used single particle cryo-electron microscopy to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag structures. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10-12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide evidence for a novel stabilization interface mediated by the HIV-2 CACTD that delivers important clues for HIV Gag lattice stabilization as well as important insights into virus maturation.

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.