Abstract

Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Env’s gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors up to 9-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 1.5-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function.

Highlights

  • Entry receptors are a major determinant of the host cell specificity of viruses [1]

  • N-terminal heptad repeat (NHR)/C-terminal heptad repeat (CHR) mutations identified by deep mutational scanning that enhanced entry increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states

  • We identify a suite of novel mutations in Env’s gp41 subunit that increase HIV-1 infection of cells expressing macaque CD4 and CCR5 receptors

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Summary

Introduction

Entry receptors are a major determinant of the host cell specificity of viruses [1]. In the case of HIV-1, human T lymphocytes, monocytes and macrophages are the principal host cells due to their expression of the HIV-1 receptor, CD4, and preferred co-receptor, CCR5. Variation in receptor orthologs among species determines the range of organisms susceptible to viral infection [2]. For HIV-1, the human CD4 receptor permits efficient infection, CD4 receptors from other nonhuman primates (NHPs) are suboptimal, and only weakly support viral entry [3,4,5,6]. The Rhesus macaque has been the favored NHP used to date, with SIV/HIV-1 hybrid viruses (SHIVs), which encode HIV-1 Env in place of SIV Env, used to model HIV-1 infection. As SHIVs encoding Envs from circulating HIV-1 strains replicate poorly

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