Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages

Abstract

Genome sequences of transmitted/founder (T/F) HIV-1 have been inferred by analyzing single genome amplicons of acute infection plasma viral RNA in the context of a mathematical model of random virus evolution; however, few of these T/F sequences have been molecularly cloned and biologically characterized. Here, we describe the derivation and biological analysis of ten infectious molecular clones, each representing a T/F genome responsible for productive HIV-1 clade B clinical infection. Each of the T/F viruses primarily utilized the CCR5 coreceptor for entry and replicated efficiently in primary human CD4(+) T lymphocytes. This result supports the conclusion that single genome amplification-derived sequences from acute infection allow for the inference of T/F viral genomes that are consistently replication competent. Studies with monocyte-derived macrophages (MDM) demonstrated various levels of replication among the T/F viruses. Although all T/F viruses replicated in MDM, the overall replication efficiency was significantly lower compared to prototypic "highly macrophage-tropic" virus strains. This phenotype was transferable by expressing the env genes in an isogenic proviral DNA backbone, indicating that T/F virus macrophage tropism mapped to Env. Furthermore, significantly higher concentrations of soluble CD4 were required to inhibit T/F virus infection compared to prototypic macrophage-tropic virus strains. Our findings suggest that the acquisition of clinical HIV-1 subtype B infection occurs by mucosal exposure to virus that is not highly macrophage tropic and that the generation and initial biological characterization of 10 clade B T/F infectious molecular clones provides new opportunities to probe virus-host interactions involved in HIV-1 transmission.

Fig 1

Highlighter analysis of near-full-length HIV-1 genomes cloned from 9-kb SG amplicon. (A) The SG amplicon is illustrated in relation to the proviral genome (top) and the viral RNA genome (middle). Gray shading indicates the regions corresponding to the two HXB2 primers, each 30 nt in length, used to generate SG amplicons. (B) Highlighter analysis of SG amplicon sequences from the vRNA/cDNA of subject WITO. The T/F sequence (consensus, CON) is indicated as a line at the top of the plot. The sequences of 9 SG amplicons are shown with nucleotide differences indicated by colored tick marks. Dark blue ticks indicate sites with mixed bases in sequencing chromatograms, following the International Union of Pure and Applied Chemistry (IUPAC) nomenclature. (C) Highlighter analyses of the near-full-length genomes cloned from the SG amplicon WITO SGA_E1. The E1 sequence was identical to the inferred T/F genome and was selected for cloning. (D to F) Highlighter analyses of the near-full-length genomes cloned from CH040 (D), CH058 (E), and CH077 (F) SG amplicons. Each Highlighter plot depicts the T/F sequence at the top and the SG amplicon that was selected for cloning. The SG amplicon sequences start at nucleotide position 582 in the 5′ LTR (U5 region) and extend to position 9606 in the 3′LTR (R region), based on the HXB2 numbering system. Thus, the scale bar at the bottom of the highlighter plots (C to F) starts at nucleotide position 1 as the first nucleotide of U3.

Fig 2

Virus production and relative infectivity of T/F viruses. PBMC were infected for 4 h at an MOI of 1 (TZM-bl IU) with transfection-derived IMC virus stocks. The cells were washed, and after 5 days the culture supernatants were collected, clarified by centrifugation, and cryostored. The samples were analyzed for p24 antigen concentration (A) and infectivity on TZM-bl cells relative to 1 ng of p24 (IU/ng-p24) (B).

Fig 3

Analysis of T/F virus coreceptor usage. TZM-bl cells preincubated for 1 h with 1.2 μM AMD3100, 10 μM TAK-779, or both were infected with 2 × 10³ TZM-bl IU (MOI = 0.24) of each virus. After a 48-h incubation period, the cells were analyzed for firefly luciferase expression. The data are plotted as the percent infection relative to infected cells not preincubated with TAK-779 or AMD3100.

Fig 4

T/F virus replication in CD4⁺ T lymphocytes and MDM. CD4⁺ T lymphocytes (A) and autologous MDM (C) were incubated overnight with 5 × 10⁴ TZM-bl IU of the control viruses YU-2, ADA, and BaL (labeled as M-tropic) and a panel of T/F viruses. Cell cultures were washed three times on day 1 and resuspended in 1 ml of fresh medium. On days 4, 7, and 10, the culture medium was completely removed for analysis of p24 antigen, and fresh medium was added back. Scatter plots show p24 antigen concentrations on day 10 for CD4⁺ T lymphocyte (B) and MDM (D) cultures. The median value for each group is indicated by a horizontal bar. Statistical significance of difference between medians was determined by calculating an exact two-tailed P value (Wilcoxon rank-sum test) and is indicated by “**”. NS, not significant.

Fig 5

T/F virus replication in CD4⁺ T lymphocytes and MDM. CD4⁺ T lymphocytes (A) and autologous MDM (C) were incubated overnight with 50 ng of p24 of the control viruses with high MRC, YU-2, ADA, and BaL (labeled as M-tropic), the low/non-mac-tropic control virus JRCSF, and a panel of T/F viruses. Cell cultures were washed three times on day 1 and resuspended in 2 ml of fresh medium. On days 4, 7, and 10, the culture medium was completely removed for analysis of p24 antigen, and fresh medium was added back. Scatter plots show the p24 antigen concentrations on day 10 for CD4⁺ T lymphocyte (B) and MDM (D) cultures. The median value for each group is indicated by a horizontal bar. Statistical significance of difference between medians of the T/F and M-tropic group was determined by calculating an exact two-tailed P value (Wilcoxon rank-sum test) and is indicated by “*”. NS, not significant.

Fig 6

Sensitivity of T/F viruses to sCD4. T/F and control viruses were preincubated with serial dilutions of sCD4 for 1 h prior to infection. The virus/sCD4 mixtures were then placed on TZM-bl cells (MOI of 0.25) in the presence of DEAE-dextran (40 μg/ml) and incubated for 48 h. The cells were then lysed and analyzed for firefly luciferase expression. (A) Bar graph representing the IC₅₀s for the different viruses analyzed. (B) Scatter plot illustrating the differences in IC₅₀s between the T/F viruses and the control viruses with high MRC (labeled as M-tropic). The median IC₅₀ for each group is indicated by a horizontal bar; the associated exact two-tailed P value (Wilcoxon rank-sum test) is shown; significance is indicated by “*”.

Fig 7

Low replication of T/F viruses in macrophage maps to the Env ectodomain. (A) MDM were infected (day 0) at an MOI of 5 (TZM-bl IU) with transfection-derived Env-IMC-LucR viruses expressing either T/F env's from CH040.c, CH058.c, CH106.c, SUMA.c, REJO.c, and THRO.c or control env's from BaL and SF162. The cells were washed, and harvested on days 1, 2, and 6 for analysis of LucR expression. (B) The scatter plot compares virus replication (RLU values) for both virus groups on day 6 (median RLU values are indicated by horizontal bars; the associated exact two-tailed P value [Wilcoxon rank-sum test] is shown). (C) Using a subset of Env-IMC-LucR viruses that expressed env's from WITO.c (♦), CH077.t (■), CH040.c (▼), CH058.c (▲), BaL (□), and SF162 (○), respectively, MDM from seven different donors were infected and analyzed for LucR expression 5 to 7 days postinfection. Scatter plots compare LucR expression in the T/F and control Env-IMC-LucR virus groups for each donor MDM (indicated by numbers 1 through 7).

Fig 8

Analysis of viral transcripts in Env-IMC-LucR virus-infected MDM. MDM were infected for 4 h at an MOI of 5 (TZM-bl IU) with transfection-derived Env-IMC-LucR viruses expressing the control env BaL or the T/F env's from CH040.c and CH077.c. The cells were washed after a 4-h incubation and then cultured for 3 days. (A) At 3 days postinoculation, the MDM were washed and analyzed for early (R-U5) viral cDNA products. A 5-fold serial dilution containing known amounts of viral DNA was analyzed in parallel. Prior to amplification, samples were DpnI digested to eliminate potential carryover plasmid DNA. (B) MDM were analyzed in parallel for LucR expression.

Fig 9

Analysis of T/F virus cDNA synthesis in MDM. MDM were infected for 8 h at an MOI of 5 (TZM-bl IU) with PBMC-derived full-length control and T/F viruses. Due to an overall lower titer of RHPA virus stocks, MDM were infected with an MOI of 2 with RHPA.c. After 8 h, the MDM were washed five times, and fresh medium was added back to each well. (A) At 24 h postinfection, the MDM were washed three times and analyzed for early (R-U5) viral cDNA products. Prior to amplification, samples were DpnI digested to eliminate any potential carryover plasmid DNA. (B) Viral cDNA products were quantified and normalized to the cDNA products of the GAPDH housekeeping gene. The ratio of viral transcripts to transcripts of GAPDH is shown.