Subunit-specific analysis of the human immunodeficiency virus type 1 reverse transcriptase in vivo

Abstract

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.

FIG. 1.

Analysis of RT subunit function by trans-complementation. (A) Illustration of the vpr-p51/p66 expression plasmid. The vpr-p51/p66 expression plasmid was constructed to allow independent expression and subunit-specific analysis of p51 and p66. The vpr-p51/p66 expression plasmid was used to construct various p51 and p66 mutants. Unless otherwise indicated, this was accomplished by inserting p51 or p66 DNA fragments at the BglII-MluI or XmaI-XhoI sites, respectively. (B) The M7 proviral clone. M7 contains a TAA stop codon at the first amino acid positions of RT and IN, three stop codons, 441 (TAA), 444 (TGA), and 447 (TAG), in the RNase H domain, a −1 frameshift at amino acid position 14 of RT, and the RNase H catalytic site mutation, D443N. (C) Model for trans expression and packaging of heterodimeric RT. Cells were cotransfected with the M7 and vpr-p51/p66 expression plasmids. Vpr-p51 incorporates p66 through interaction and stable association of the two subunits (Vpr-p51 and p66) within the cellular cytoplasm. Specific interaction between Vpr and Pr55^Gag leads to the incorporation of the Vpr-p51/p66 complex into progeny virions. Subsequent cleavage by the viral PR generates wild-type RT heterodimer (p51/p66).

FIG. 2.

Vpr-p51-mediated p66 virion incorporation and proteolytic processing. M7 proviral DNA was transfected alone or together with the vpr-IN expression plasmid and either the vpr-p66, vpr-p51/p66, or vprΔp51/p66 expression plasmid. Wild-type SG3 was included as a control. Transfection-derived virions were concentrated by ultracentrifugation, lysed, and analyzed by immunoblotting for p51/p66 (A), p66 (B), or p24 (C) as described in Materials and Methods.

FIG. 3.

trans-complementation analysis of RT-IN-deficient virus. (A) Analysis of infectivity. Transfection-derived viruses were analyzed for infectivity using the TZM-bl reporter cell line as described in Materials and Methods. Infectivity is expressed as a percentage of the wild-type virus control. Viruses were derived from 293T cells with (+) or without (−) cotransfection of vpr-IN. The results of three independent experiments are shown. (B) Analysis of heterodimeric trans-RT activity. Increasing DNA concentrations of vpr-p51/p66 (ranging from 0.5 to 3.0 μg) were transfected into 293T cells together with a constant amount of M7 and vpr-IN. Culture supernatants were collected and treated as described in Materials and Methods. The transfection-derived virions were analyzed for infectivity using the TZM-bl reporter cell line (black bars) and for RT activity using the chemiluminescent RT assay (Roche) (white bars). Results are expressed as a percentage relative to an equal amount of wild-type SG3 virus.

FIG. 4.

Subunit-specific analysis of the YMDD aspartates. The wild-type, control, and mutated vpr-p51 expression plasmids were cotransfected into 293T cells with the M7 and vpr-IN DNAs to generate virions. (A) Analysis of infectivity. Infectivity, expressed as a percentage of wild-type virus, was analyzed from three independent experiments. (B and C) Analysis of viral DNA synthesis. DNA products of reverse transcription were analyzed as described in Materials and Methods. Early (R-U5) (B) and late (R-gag) (C) DNA products were amplified by PCR, resolved on 1.0% agarose gels, and stained with ethidium bromide. To approximate the relative amount of each of the amplified DNA products, 10-fold serial dilutions of pSG3 DNA (ranging from 10¹ to 10⁵ copies) were prepared and analyzed in parallel. Distilled water (dw) was included as a negative control.

FIG. 5.

Analysis of p66 virion incorporation. M7 proviral DNA was cotransfected into 293T cells with the vpr-p51/p66 plasmids containing various mutations in the YMDD aspartates. The transfection-derived virions were concentrated by ultracentrifugation, lysed, and examined for p66 (A), p51 (B), or p24 (C) by immunoblot analysis as described in Materials and Methods.

FIG. 6.

Putative interactions of the p51 D185/D186 aspartates, including interactions of the p51 D185/D186 (yellow) of HIV-1 RT at the junction of the p51 palm (cyan and yellow), p51 connection (white), and p51 fingers (green) subdomains in the structure of the RT/DNA/dNTP complex (PDB code 1RTD). The Trp-rich region is shown at the interface of the p51 (white) and p66 (red) subunits and proximal to the DNA binding cleft (red).

FIG. 7.

Analysis of putative interactions of p51 D185/D186. Virions containing p51 mutations at amino acid residues within interacting distance of its YMDD aspartates were analyzed for infectivity on TZM-bl reporter cells as described in Materials and Methods. Infectivity is expressed as a percentage of the wild-type virus control. The results of five independent experiments are shown.