Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription

Abstract

During the early stages of HIV-1 replication the conical capsid composed of p24(CA) protein dissociates from the rest of the cytoplasmic viral complex by a process called uncoating. Although proper uncoating is known to be required for HIV-1 infection, many questions remain about the timing and factors involved in the process. Here we have used two complementary assays to study the process of uncoating in HIV-1-infected cells, specifically looking at the timing of uncoating and its relationship to reverse transcription. We developed a fluorescent microscopy-based uncoating assay that detects the association of p24(CA) with HIV-1 viral complexes in cells. We also used an owl monkey kidney (OMK) cell assay that is based on timed TRIM-CypA-mediated restriction of HIV-1 replication. Results from both assays indicate that uncoating is initiated within 1 h of viral fusion. In addition, treatment with the reverse transcriptase inhibitor nevirapine delayed uncoating in both assays. Analysis of reverse transcription products in OMK cells revealed that the generation of early reverse transcription products coincides with the timing of uncoating in these assays. Collectively, these results suggest that some aspect of reverse transcription has the ability to influence the kinetics of uncoating.

Fig. 1.

Results from the in situ uncoating assay. (A) Top Left: One Z-section from a 1 h time point. The outlined box is enlarged in the other three panels to show S15-mCherry, GFP-Vpr, and p24^CA signals. Fused virions are punctate signals that are GFP positive and mCherry negative, and were classified as associated with p24^CA (horizontal arrows) or not associated with p24^CA (vertical arrows). (B) Number of coated and uncoated fused virions counted at 1, 2, and 4 h post-infection. Above each bar is the percentage of coated viral particles and the total number of virions counted. (C) In situ uncoating assay was conducted on wild-type and p24^CA mutants. Graphed is the percentage of coated viral particles at each time point. Error bars denote SE among three independent experiments. (D) In situ uncoating assay was conducted in the presence or absence of nevirapine (NVP). Graphed is the percentage of coated viral particles at each time point. Error bars denote the SE among two independent experiments.

Fig. 2.

Results from the CsA washout assay. (A) CsA washout assay was conducted with or without a 2 h nevirapine (NVP) treatment. Graphed is the percentage of GFP-positive cells at each time of CsA washout normalized by setting the percentage at 4 h to 100%. Shown is a representative experiment from eight independent experiments. Error bars denote SE. (B) CsA washout assay was conducted using HIV-GFP or HIV-GFPNevR with or without a 2 h nevirapine treatment. Shown are normalized data of a representative experiment from four independent experiments. Error bars denote SE. (C) CsA washout assay was conducted using HIV-GFP without nevirapine treatment or with nevirapine treatment for 2, 3, or 4 h. Shown are normalized data of a representative experiment from four independent experiments. Error bars denote SE. (D) CsA washout assay was conducted using R7X4-GFP with or without a 2 h nevirapine treatment. Shown are normalized data of a representative experiment from three independent experiments. Error bars denote SE.

Fig. 3.

Viral fusion in OMK cells. (A) Ammonium chloride add-in assay was conducted to examine VSV-g–mediated viral fusion in OMK cells. (B) AMD3100 add-in assay was conducted to examine HIV Env-mediated viral fusion in OMK cells. For both A and B, controls were continuous drug treatment and no treatment. Shown is a representative experiment from three independent experiments. Error bars denote SE.

Fig. 4.

Assays to correlate reverse transcription with uncoating. (A) Nevirapine (NVP) add-in assay was performed in OMK cells. Controls were continuous treatment with nevirapine and no treatment. Shown is a representative experiment from three independent experiments. Error bars denote SE. (B) Real-time PCR analysis was conducted on OMK cells infected with HIV-GFP. Controls were ethanol treatment, nevirapine treatment, and mock infection. Shown is a representative experiment from three independent experiments. Error bars denote SE among three reactions.