CXCR4- and CCR5-Tropic HIV-1 Clones Are Both Tractable to Grow in Rhesus Macaques

Abstract

A major issue for present HIV-1 research is to establish model systems that reflect or mimic viral replication and pathogenesis actually observed in infected humans. To this end, various strategies using macaques as infection targets have long been pursued. In particular, experimental infections of rhesus macaques by HIV-1 derivatives have been believed to be best suited, if practicable, for studies on interaction of HIV-1 and humans under various circumstances. Recently, through in vitro genetic manipulations and viral cell-adaptations, we have successfully generated a series of HIV-1 derivatives with CXCR4-tropism or CCR5-tropism that grow in macaque cells to various degrees. Of these viruses, those with best replicative potentials can grow comparably with a pathogenic SIVmac in macaque cells by counteracting major restriction factors TRIM5, APOBEC3, and tetherin proteins. In this study, rhesus macaques were challenged with CXCR4-tropic (MN4/LSDQgtu) or CCR5-tropic (gtu + A4CI1) virus. The two viruses were found to productively infect rhesus macaques, being rhesus macaque-tropic HIV-1 (HIV-1rmt). However, plasma viral RNA was reduced to be an undetectable level in infected macaques at 5-6 weeks post-infection and thereafter. While replicated similarly well in rhesus peripheral blood mononuclear cells, MN4/LSDQgtu grew much better than gtu + A4CI1 in the animals. To the best of our knowledge, this is the first report demonstrating that HIV-1 derivatives (variants) grow in rhesus macaques. These viruses certainly constitute firm bases for generating HIV-1rmt clones pathogenic for rhesus monkeys, albeit they grow more poorly than pathogenic SIVmac and SHIV clones reported to date.

Keywords: CCR5-tropic; CXCR4-tropic; HIV-1; HIV-1rmt; primate model; rhesus macaque.

FIGURE 1

Basic genome structure of HIV-1rmt clones. The three HIV-1rmt clones indicated have been constructed from three distinct primate immunodeficiency viruses as shown. Genomic regions of HIV-1rmt clones derived from HIV-1 NL4-3, SIVmac MA239, and SIVgsn 166 (SIV isolated from the greater spot-nosed monkey) are depicted by white, blue, and orange areas, respectively. Generation and characterization of HIV-1 NL4-3 (Adachi et al., 1986), SIVmac MA239 (Shibata et al., 1991), CXCR4-tropic HIV-1rmt designated MN4/LSDQgtu (Nomaguchi et al., 2013b; Nomaguchi et al., 2017), CCR5-tropic HIV-1rmt designated MN5/LSDQgtu (Nomaguchi et al., 2013b; Nomaguchi et al., 2017), and CCR5-tropic HIV-1rmt designated gtu + A4CI1 (Doi et al., 2017) have been fully described previously. MN4/LSDQgtu and MN5/LSDQgtu carry growth-enhancing mutations in Gag-capsid, Pol-integrase, and Env regions as previously described (Nomaguchi et al., 2013a,b). GenBank accession numbers for sequences of NL4-3, MA239, 166, MN4/LSDQgtu, and MN5/LSDQgtu are AF324493, M33262, AF468659, LC315178, and LC315179, respectively.

FIGURE 2

Growth property of HIV-1rmt clones in rhesus PBMCs and individuals. (A) Viral replication kinetics in rhesus PBMCs infected with CXCR4-tropic MN4/LSDQgtu, CCR5-tropic MN5/LSDQgtu, or CCR5-tropic gtu + A4CI1. PBMCs were prepared from rhesus macaques MM630 and MM631, and spin-infected with cell-free viruses obtained from transfected 293T cells as previously described (Nomaguchi et al., 2013b, 2014). Cell numbers and input viral amounts used were 2.0 × 10⁶ and 4.1 × 10⁶ RT units, respectively. (B) Kinetics of plasma viral loads in rhesus macaques inoculated with CXCR4-tropic MN4/LSDQgtu or CCR5-tropic gtu + A4CI1. Rhesus macaques MM581, MM602, and MM631 were infected with cell-free viruses obtained from transfected 293T cells, and monitored for viral RNAs in plasma as previously described (Otsuki et al., 2014; Ishida et al., 2016). MM581 and MM602 were inoculated intravenously with 4.3 × 10⁵ TCID₅₀ of MN4/LSDQgtu as determined in a macaque cell line HSC-F (Akari et al., 1999). MM631 was inoculated with gtu + A4CI1 intravenously (5.0 × 10⁶ TCID₅₀ in HSC-F cells) and intraperitoneally (1.5 × 10⁷ TCID₅₀ in HSC-F cells). Infection experiments (MM581/MM602 and MM631) were separately and independently conducted, and the detection limits for the MM581/MM602 and MM631 experiments were 250 and 500 copies/ml, respectively. The TRIM5 genotypes as analyzed by the previously described method (Wilson et al., 2008) for MM581, MM602, MM630, and MM631 are TRIM5^TFP/TFP (Mamu-1/Mamu-3), TRIM5^TFP/TFP (Mamu-3/Mamu-3), TRIM5^TFP/TFP (Mamu-3/Mamu-3), and TRIM5^Q/Q (Mamu-4/Mamu-4), respectively.