Highlights d HIV cure studies rely on precise HIV reservoir assays that can be done at scale d Multiplexed droplet digital PCR can probe multiple HIV genome targets per sample d HIV-1 proviruses containing 5 probed genomic regions are likely intact d PCR-based counting of T cells improves reservoir quantification in tissues

High-throughput HIV reservoir assays for diverse HIV subtypes are needed Cross-subtype intact proviral DNA assay (CS-IPDA) works across global HIV-1 subtypes CS-IPDA can be used in cure studies in Africa and Asia where HIV prevalence is high CS-IPDA on samples from Kenyan infants on ART show expected decay of intact HIV DNA

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.Viruses 2020, 12, 241 2 of 20 in macaque cells [7], most studies employing the SHIV/macaque model have used HIV-1 Envs that were lab adapted to enhance macaque infection, and/or that were derived from HIV-1 variants isolated from the chronic stages of infection [7]. These evolved and chronic-stage Envs are highly pathogenic and transmissible in macaques [8]. While these characteristics enabled robust infection of macaques, evolved and chronic-stage Envs display altered structural conformations and increased susceptibility to neutralizing antibodies compared to Envs from circulating HIV-1 strains [8,9]. Use of these Envs in SHIVs therefore compromises the relevance of the SHIV/macaque model for informing HIV-1 infection in humans [7].In 2012, it was discovered that a single amino acid mutation in macaque CD4 was responsible for the poor ability of HIV-1 to utilize macaque receptors for entry [3]. In vitro evolution experiments also revealed that single amino acid substitutions at sites 204 or 312 in Env's gp120 subunit allow HIV-1 to more effectively use macaque CD4 as an entry receptor [4]. These mutations disrupted quaternary contacts between Env trimer subunits, and similar to Envs encoded by highly pathogenic SHIVs, increased sensitivity to neutralizing antibodies [10].Identification of these mutants prompted further efforts to identify additional Env mutations that would enhance usage of macaque CD4. In 2016, L...

8Although Rhesus macaques are an important animal model for HIV-1 vaccine development 9research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic 10 determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 11 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. 12 Recent research efforts employing either laboratory evolution or structure-guided design 13 strategies have uncovered several mutations in Env's gp120 subunit that enhance binding of 14 macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env 15 mutations that promote infection of macaque cells, we utilized deep mutational scanning to 16 screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque 17 receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad 18 repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells 19 bearing macaque receptors by up to 38-fold. Many of these mutations also modestly increased 20 infection of cells bearing human CD4 and CCR5 (up to 13-fold). NHR/CHR mutations identified 21 by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing 22 antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that 23 they promote sampling of an intermediate trimer conformation between closed and receptor 24 bound states. Identification of this set of mutations can inform future macaque model studies, 25 and also further our understanding of the relationship between Env structure and function. 263 Importance 27Although Rhesus macaques are the favored non-human primate animal model used in HIV-1 28 research, most circulating HIV-1 strains poorly infect macaque cells. Studies using macaques to 29 model HIV-1 infection often use evolved, or mutant HIV-1 variants that are able to utilize 30 macaque CD4, but these HIV-1 variants poorly model infection by circulating strains. In this 31 work, we sought to identity HIV-1 mutations that would allow entry into macaque cells, but 32 that would maintain critical characteristics of circulating HIV-1 strains. We employed a powerful 33 experimental method to simultaneously assess the effects of thousands of individual HIV-1 34 mutations on infection of cells bearing macaque receptors. We identified many previously 35 uncharacterized mutations that enhance infection of circulating HIV-1 strains into cells bearing 36 macaque receptors by up to 38-fold. Identification of these mutations may be of use in future 37 macaque model studies. 38Env, used to model HIV-1 infection. As SHIVs encoding Envs from circulating HIV-1 strains 51 replicate poorly in macaque cells (7), most studies employing the SHIV/macaque model have 52 used HIV-1 Envs that were lab adapted to enhance macaque infection, and/or that were 53 derived from HIV-1 variants isolated from the chronic stages of infection (7). These evolved and 54 chronic s...

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