The HIV-1 capsid serves as a nanoscale reaction vessel for reverse transcription

Abstract

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Fig 1. IP6-dependent ERT reactions are resistant to degradation by DNase I.

ERT reactions were performed with HIV-1 virions in the presence of the indicated concentrations of IP6, with or without added DNase I. Reactions were incubated for 4h. In reactions 10–12, PF74 was added to a concentration of 20 μM and DNAse I to 20 μg/ml, and the reactions were incubated for an additional 60 min. Products were purified and quantified by qPCR using primers specific for minus strand strong stop (MSS) and full length minus strand (FLM) amplicons. Results shown are representative of three independent experiments. Panel B shows the compiled results of the effects of DNase I treatment on ERT reactions (containing 10 μM IP6) from four independent experiments, with plasmid DNA included in each experiment as a control for DNase I activity.

Fig 2. Reverse transcription in nonpermeabilized HIV-1 virions does not require addition of IP6, is resistant to PF74, and is resistant to DNase I.

Reactions were incubated for 14h. A. Reactions with wild type HIV-1 virions were performed in the presence or absence of dNTPs (dATP, dCTP, dGTP, and TTP; 0.1 mM each), 0.1% Triton X-100, and with or without 10 μM IP6. The right panel shows compiled results from four independent NERT experiments, three of which also contained ERT reactions. B. Levels of inhibition observed in reactions performed in the absence or presence of detergent and IP6 (NERT and ERT, respectively) by the indicated inhibitors: 10 μM azidothymidine triphosphate (AZTTP); 1 μM efavirenz (EFV); 10 μM stavudine triphosphate (d4TTP); 1 mM aldrithiol (AT-2); 10 μM PF-3450074 (PF74). Shown are mean values and standard deviations calculated from results of three independent experiments. C. Effects of DNAse I treatment of nonpermeabilized reactions. Left panel: quantification of DNA products in reactions that were treated with DNase I (20 μg/ml) for 1h following the 14h reaction. Right panel: parallel control reactions containing plasmid DNA. Shown are the results from four independent experiments. On average, DNAse I reduced the viral DNA products in reverse transcription reactions by 20% and plasmid DNA by 97%.

Fig 3. Reverse transcription in nonpermeabilized HIV-1 virions requires the gp41 CT.

A. Reactions containing wild type (WT), Env-deficient (Env^-), and the indicated gp41 C-terminal truncation mutants were performed in the absence (NERT) and presence (ERT) of detergent and IP6. Reactions were incubated for 14h and the early (MSS) and late (FLM) products quantified by qPCR. Numerical values in the graph represent the NERT to ERT ratio of late (FLM) reverse transcription product levels in each reaction. B. Mean values of NERT/ERT ratios determined from five independent experiments. Error bars depict standard deviations. Asterisks indicate significant differences between the indicated viruses and Env- mutant (95% confidence interval) obtained with the paired ratio T test in Graphpad Prism. C. Pelleted virions used in the reactions shown in A were analyzed by immunoblotting using a monoclonal antibody recognizing a membrane-proximal epitope in the gp41 CT. D. Ratio of band intensities of gp41 and CA shown in B. Error bars represent the range of values from two technical replicates.

Fig 4. NERT activity depends on the viral Env protein.

NERT and ERT reactions were performed with the indicated viruses. A. Analysis of HIV-1 virions produced by cotransfection of Env- provirus with: HIV-1HXB2 Env expression plasmid (HIV-1); A-MLV-pseudotyped HIV-1 virions (A-MLV); and VSV-pseudotyped HIV-1 virions (VSV-G). Numerical values in the graph represent the NERT to ERT ratio of late (FLM) reverse transcription product levels in each reaction. The right panel shows mean values and standard deviations from five independent experiments. The A-MLV and VSV-G pseudotyped virions did not exhibit statistically significantly higher values than Env- virions. B. Analysis of wild type and Env- SIV_mac239 virions. Numerical values in the graph are as in panel A. The right panel shows mean values and standard deviations from four independent experiments. Significance (95% confidence interval) was analyzed with the paired t test in Graphpad Prism. C. Analysis of wild type (NL4-3), Env-, and NL4-3 chimerae encoding Env proteins from HIV-1 primary isolates. Shown are the levels of late product synthesis detected in each NERT and ERT reaction. Numerical values in the graph are as in panel A. Right panel: mean values obtained from three independent experiments. Asterisks designate significant differences between the indicated virions and Env- virions.

Fig 5. NERT efficiency in HIV-1 virions varies with the cellular source of the virions.

Wild type and Env- HIV-1 virions were harvested from the indicated T cell lines, concentrated, and assayed for NERT and ERT activity. A. Shown are the levels of early (MSS) and late (FLM) products from a representative experiment. The numerical values above each sample represent the ratio of late product levels in the corresponding NERT and ERT reactions. B. Shown are the mean NERT/ERT ratios (FLM products) for wild type and Env- particles released from the indicated T cell lines from five independent experiments. Asterisks show statistically significant NERT activity levels vs. the corresponding Env- particles using the ratio paired t test.

Fig 6. Minimally processed virus stocks also exhibit NERT.

NERT reactions were performed with minimally processed wild type (WT) and Env- virions freshly harvested from transfected 293T cells and not subjected to filtration, concentration, or freezing. Shown are the levels of early and late product synthesis in two separate preparations of wild type and Env- virions. Values represent the calculated stoichiometry of the reactions based on theoretical virion concentrations calculated from p24 ELISA data. Results are representative of two independent experiments.

Fig 7. HIV-1 virions with cleavable gp41 proteins exhibit reduced NERT activity.

A. NERT and ERT reactions were performed with the indicated wild type, Env-, and CT point mutant virions. Shown are the early (MSS and FST) and late (FLM and SST) products from the corresponding NERT and ERT reactions. B. Ratios of the DNA levels detected in the NERT and ERT reactions shown in A. C. Immunoblot analysis of the concentrated virions used in this experiment. D. Mean NERT/ERT values and standard deviations determined from six independent experiments. Asterisks represent significance (95% confidence interval) determined using ratio paired t test (Graphpad Prism).

Fig 8. NERT activity can occur in virions lacking a mature capsid.

A. NERT and ERT assays of the indicated wild type (NL4-3) and Gag cleavage mutants. As depicted in S1 Fig, CA5 virions contain uncleaved CA-SP1 protein; CA6 virions contain uncleaved CA-SP1-NC; MA-CA: uncleaved MA-CA protein; MA-p2: uncleaved MA-CA-SP1; MA-NC: uncleaved MA-CA-SP1-NC; and MA-p6: uncleaved MA-CA-SP1-NC-SP1-p6. Shown are the early and late product DNA levels (FST and FLM, respectively). Numerical values shown represent the relative efficiency of ERT (FLM products normalized by exogenous RT activity added to the reactions). B. Mean values and standard deviations of NERT values, normalized by exogenous RT in the virus preparations, from five independent experiments. Asterisks indicate mutants that exhibited significant different NERT activity vs. wild type HIV-1 virions (95% CI; ratio paired T test). C and D. HIV-1 mutants bearing large deletions in CA are competent for NERT. Two mutants lacking nearly the entire N-terminal domain of CA were assayed for NERT and ERT alongside wild type and Env- virions. The results shown are from one of four independent experiments. D. Mean values and standard deviations of NERT values, normalized by exogenous RT in the virus preparations, from four independent experiments. Asterisks indicate mutants that exhibited significant different NERT activity vs. wild type HIV-1 virions (95% CI; ratio paired T test).