Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1

Abstract

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.

Fig. 1.

Infection of pig-tail cells with HIV-1 and restriction by TRIM5 isoforms. (A) PBMC costimulated with anti-CD3 and anti-CD28 from rhesus and pig-tailed macaques were transduced with HIV-GFP virus pseudotyped with VSV-G. The cells were obtained from three experimental donor animals of each species, as indicated by three different columns (black, white, and gray). After costimulation, >98% of the cells were T cells. (B) Transduction of MAGI indicator cells expressing human (Hu) or rhesus (Rh) TRIM5α or pig-tail (Pt) TRIM5η or control cells with increasing amounts of HIV-GFP pseudotyped with VSV-G. Infections were performed in duplicate and the average is shown. One of three representative experiments is shown. The inset shows a Western blot for flag-tagged TRIM5 proteins. (C) Transduction of control cells or cells expressing owl monkey (OWM) or pig-tail TRIM-Cyp with HIV-1_NL4-3-luc/VSV-G. Infections were performed in triplicate. The average level of infection relative to the level of infection of the control cells is shown.

Fig. 2.

Replication of the HSIV-vif chimera. (A) Schematic diagram comparing the genomic structures of HIV-1_NL4-3, HSIV-vif, and SIVmne027. (B) Replication of HIV-1_NL4-3 and HSIV-vif in anti-CD3/anti-CD28-costimulated human PBMC. (C) Replication of HSIV-vif in comparison to SIVmne027 and HIV-1_NL4-3 (NL4-3) in anti-CD3/anti-CD28-costimulated Pt PBMC. Duplicate infections were carried out at an MOI of 0.01. Replication was monitored by determining the amount of infectious virus produced per ml at 3- to 4-day intervals postinfection using TZM-bl reporter cells. Infections of two different macaque donors are shown and were performed using independently derived stocks of virus.

Fig. 3.

Replication of different HIV-1 vif chimeras. The replication kinetics of two additional HIV-1 chimeras with minimal vif (HSIV-vif-Yu2 and HSIV-vif-AD8) substitutions are shown in comparison to the HSIV-vif clone based on HIV-1_NL4-3. The experiments were performed as described for Fig. 2D.

Fig. 4.

Infection of juvenile pig-tailed macaques. Two juvenile pig-tails were inoculated intravenously with HSIV-vif based on HIV-1_NL4-3. (A) Plasma viral load measurements; (B) CD3⁺ CD4⁺ T-cell counts in peripheral blood; (C) HIV-1 antibody titer.

Fig. 5.

Infection of newborn pig-tailed macaques. Two newborn pig-tails were inoculated intravenously with HSIV-vif based on HIV-1_NL4-3. (A) Plasma viral loads; (B) CD3⁺ CD4⁺ T-cell counts in peripheral blood; (C) HIV-1 antibody titer.

Fig. 6.

Summary of mutations in the env su gene. env su sequences were cloned from PBMC DNA isolated 72 weeks postinoculation from both juvenile pig-tailed macaques. Mutations relative to the HIV-1_NL4-3 env su sequence are shown. Base number 0 is position 178 in the NL4-3 env sequence. The color code for nucleotide base changes and mutation symbols are provided on the vertical axis. Alignment and highlighted mutations were generated by using Highlighter (http://www.hiv.lanl.gov/content/sequence/highlight/highlighter.html).

Fig. 7.

Vif start site mutations and degradation of Pt APOBEC3 proteins. (A) Diagram of the sequences between the HIV-1 vif gene start site and SIV vif gene start site. In HSIV-vif, each ATG within the HIV-1 vif sequence was changed to ACG. Reversion mutations observed in the HIV-1 vif met codons located 5′ to the SIV vif start site at 32 and 72 weeks postinoculation are shown. A mutant of HSIV-vif with the HIV-1 vif met codons (HSIV-vif-3st) is also shown. (B) Vif protein expression and degradation of Pt APOBEC3 proteins by SIVmne027, HIV-1_NL4-3, NL4-3ΔVif, HSIV-vif, and HSIV-vif-3st. Each provirus was cotransfected into 293T cells with HA-tagged Pt A3G or Pt A3F. Protein expression in cell lysates or concentrated virions prepared from supernatants was determined by immunoblotting with anti-HA, anti-Gag p24 antibody, or a control antibody against β-actin. (C and D) The relative infectivity of each clone was determined by comparing infectivity in the presence or absence of Pt A3G (C) or Pt A3F (D). Infections were performed in quadruplicate, and means ± the standard errors of the mean are shown. (E) Infectivity of viruses produced from Pt PBMC. The infectivity of each virus was determined using supernatants harvested at 3 to 7 days postinfection with HSIV-vif, HIV-1_NL4-3, or SIVmne027. Infectivity is defined as the average TCID₅₀ per pg of p24 (HIV-1NL4-3 or HSIV-vif) or p27 (SIVmne027) ± the standard deviations of five independent experiments.