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. 2016 Nov 25;13(1):41.
doi: 10.1186/s12981-016-0125-8.

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections

Kendall C Krebs #  1 , Meijuan Tian #  1   2 , Mohammed Asmal  3 , Binhua Ling  4 , Kenneth Nelson  1 , Kenneth Henry  1 , Richard Gibson  1   2 , Yuejin Li  1 , Weining Han  1 , Robin J Shattock  5 , Ronald S Veazey  4 , Norman Letvin  3 , Eric J Arts  1   6   2 , Yong Gao  1   6   2
Affiliations

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections

Kendall C Krebs et al. AIDS Res Ther. .

Abstract

Background: New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques.

Results: Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans.

Conclusion: Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.

Keywords: Glycosylation; Replicative fitness; Simian–human immunodeficiency chimeric virus; Transmission.

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Figures

Fig. 1
Fig. 1
Production of pREC_nfl_SHIVenv containing the gp120 coding region derived from acute HIV-1 infections. a Construction of pREC_nfl_SHIVKB9_envB vector/URA3. The URA3 gene and the corresponding AHI env B genes only replace a region flanked by KB9 sequence (HXB2 numbering nt5996-nt8638). SHIVKB9 is a SIVmac239 virus containing only a portion of the HIV-1 env89.6 gene, i.e. from the 1st exon of tat to the end on the 2nd exon of tat (HXB2 numbering nt5823-nt8676, shadowed in yellow). b 20 acute HIV-1 subtype B envelope (Env) and three chronic HIV-1 Envs (i.e. I10, K44, and Q0) were cloned into pREC_nfl_SHIVKB9Δenv/URA3 (or pREC_nfl_HIVΔenv/URA3) by homologous yeast recombination/gap repair to produce the pREC_nfl_SHIVKB9_envB1-20 and pREC_nfl_HIVenv_B1-20 vectors
Fig. 2
Fig. 2
Chimeric viruses SHIVenv_B and HIVenv_B propagation in different PBMCs and cell lines. U87.CD4.CCR5, 174×CEM.CCR5, human PBMCs or macaque PBMCs were exposed to SHIVenv_B3, _B11, and _B20 (a), or HIVenv_B3, _B11, and _B20 (b), or SHIVenv_KB9 (c) chimeric viruses. Virus production was measure by RT activity in the supernatant over 17 days
Fig. 3
Fig. 3
Veritrop assay detecting the function of the HIV-1 envelope proteins in the context of SHIVKB9 backbone. A schematic of the Veritrop cell-to-cell fusion assay is shown in (a). The target cell, U87.CD4.CCR5 was transfected with pDM128fLuc, a vector where expression of firefly luciferase gene is dependent on both HIV-1 Rev and Tat produced by the effector cell [34]. The effector cell is transfected with pREC_nfl_SHIVKB9env_B vectors, which express entire SHIV proteome and produce virus particles that lack the proper SHIV genomic RNA for de novo replication [20]. Levels of cell fusion (via relative light units from Luciferase activity) are shown for the effector cells transfected with pREC_nfl_SHIV KB9env_B vectors (b)
Fig. 4
Fig. 4
Expression of p27 capsid and reverse transcriptase of the SHIVenv_B in the transfection supernatant from 293T cells. SIV p27 was monitored by antigen capture assay in the supernatant of 293T cells transfected with pREC_SHIVKB9env_B (B1, B3, B4, B11, B14, B17 and B20) (a). Reverse transcriptase activity was also measured using serial dilutions of the supernatant from the same transfected cells. RT activity is report as virtual TCID50 as previously described (b) [22]. The correlation of p27 and virtual TCID50 of each SHIVenv_B virus was plotted (c)
Fig. 5
Fig. 5
SHIV replication in macaques exposed to a pool of 16 SHIVenv_B viruses or just SHIVenv_B3. a The SHIVenv_B pool harboring 16 chimeric viruses were intravenously injected into two macaques of Indian origin. Infection of the two macaques was monitored by collecting plasma samples for 62 days and measuring viral RNA levels by quantitative real time RT-PCR for SIV Gag (Duke Human Vaccine Institute). Results are only shown for animal m328-08 in which SHIVenv infection was established. b The SHIVenv_B3 was used to infect three other rhesus macaques of Indian origin. Viral RNA was monitored for 140 days
Fig. 6
Fig. 6
Sequence analyses from plasma of the infected macaques. HIV RNA was extracted from plasma collected from the m328-08 and then subjected to RT-PCR amplification, TOPO cloning, and sequence analyses of 15–19 clones from days 14, 19, and 26 post inoculation. A 480 nt gp120 coding region (C2-V3) of the m328-08 clones were aligned with all the SHIVenv_B clones within the inoculating pool using CluxtalX. Neighbor joining trees were constructed using FigTree and presented in (a). Only a single SHIVenv_B3 clone established and maintained infection in macaque 328-08. The RT-PCR amplified HIV-1 gp120 product derived from ~10,000 RNA copies at day 19 was subjected to 454 pyrosequencing. The C2-V3 sequences from day 19, the 16 SHIVenv_B sequences from the inoculating pool, and a set of subtype B reference sequences were aligned using Bayesian algorithm (Beast) [73] and trees were constructed using a maximum likelihood method. The tree presented in b was drawn with FigTree v1.4 (http://tree.bio.ed.ac.uk/software/figtree)
Fig. 7
Fig. 7
Using the Affinofile system to measure entry efficiency of the HIVenv_B chimeric viruses. a The 293-Affinofile cells were treated with ponasterone A (4 and 0.125 ng/ml) to obtain high (~15,830 copies/cell) and low CCR5 surface expression (~1365 copies/cell) and with increasing concentrations of minocycline to obtain range of CD4 surface expression (1800–125,000 copies/cell). 293T cells with different CD4/CCR5 levels were exposed to each of the Env pseudotyped viruses [25] using the Env expression plasmids derived the CHAVI AHI [7] (b, c) and the NYU/Aaron Diamond AHI cohort [25] (d, e). Virus production was monitored by luciferase expression from the LucAM transgene [25]. An average maximal infection was calculated from the relative light units (RLUs) produced from infection of 293T with the highest CD4 and CCR5 surface expression. All other levels of infectivity are plotted as a percentage of the RLU obtained relative to RLU with maximal CD4 and CCR5 expression. b, c Indicate levels of infectivity at high CCR5 over a range of CD4 and d, e, low CCR5 over a range of CD4. The level of infectivity was measured and presented for all HIV-1env clones derived from AHI (b), only those HIV-1env clones from CHAVI AHI patients which were also used to construct the SHIVenv pool (c). In all four graphs, the level of infectivity with the HIV-1env B3 clone (i.e. the SHIVenv_B clone establishing infection in the macaque) is indicated with purple line. The dark blue and light blue lines are the levels of infectivity using the AHI HIV-1env clones from CHAVI and the NYU/Aaron Diamond cohort, respectively
Fig. 8
Fig. 8
Sensitivity to entry inhibitors and entry kinetics. a Each HIV-1env_B virus (CHAVI acute, NYU acute, and two chronic HIV-1 strains as control) was tested for inhibition by enfuvirtide, TAK779, and maraviroc. Inhibitory concentration for 50% inhibition (IC50) was plotted for AHI HIV-1env viruses in a. The IC50 values of HIV-1env_B3 are shown with purple symbols. b Entry kinetics was measured with the HIV-1env_B samples by performing time-of-drug inhibition experiments with the RT inhibitor, 3TC
Fig. 9
Fig. 9
The fitness difference of AHI HIV-1env chimeric virus relative to the control HIV-1env chimeric virus derived from chronic infection. As described in the schematic (a), HIV-1env_B chimeric viruses were competed against three reference viruses (i.e. HIVenv_I10, HIVenv_K44, and HIVenv_Q0) in HIV-negative PBMCs as described in the “Methods” section. a is also schematic describing the PCR and 454 pyrosequencing methods used to detect and quantify dual infection (see “Methods” section for more detail). The percent replication relative to HIVenv_I10, HIVenv_K44, and HIVenv_Q0 is shown in b. Total relative fitness (the addition of all relative fitness values) is presented in c to provide a rank order for relative fitness for the 19 HIVenv viruses tested
Fig. 10
Fig. 10
Characterization of envB3 and other env clones in SHIVenv_B pool. Comparison of putative N-linked glycosylation sites in the HIVenv_B sequences (a), in conserved region (b), in V1/V2 region (c), and in V4/V5 region (d). Net positive charge (e) and PSSM score (f) were also calculated for the HIVenv_B sequences
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