Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag-Pol are not essential for incorporation of the Pr160(gag-pol) into virus particles

Abstract

Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55(gag). Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.

FIG. 1.

HIV-1 Pr160^gag-pol mutations. (A) Mature Gag protein domains and pol-encoded p6*, PR, and RT domains. The name of each construct is shown at the left. pGAG was constructed by deletion of the pol gene. GPfs was engineered by placing the gag and pol in the same open reading frame via a five-T nucleotide deletion in the overlap region of gag-pol. The other mutants were derived from GPfs by deletion of gag-pol sequences to different extents (see Materials and Methods for specific details). The pGAG and the wt and mutant GPfs were all expressed on the HIVgpt backbone. ΔMA contains a deletion of 105 codons and a replacement of 4 codons in the MA protein. For ΔNC, sequences encoding the p2 and most of NC were deleted and replaced by two amino acid residues. Combination of the ΔMA and ΔNC mutations generated the construct Δ(MA+NC). In the construct ΔMHR, 28 codons of the CA domain (CA codon 148 to 177), covering the major homology region (codon 153 to 172) were deleted and replaced by three amino acid residues. ΔCA-1 contains a deletion of 111 codons (codon 81 to 191) and insertion of one amino acid residue into the deleted region. ΔCA-2 is identical to ΔCA-1 except that it contains a further 3′ deletion downstream of the N terminus of p2. A further 5′ deletion in ΔCA-1, deleted from CA codons 81 to 12, yielded ΔCA-3. Combination of the ΔCA-2 and ΔCA-3 mutations generated the ΔCA-4, and introduction of the ΔNC mutation to the ΔCA-4 yielded Δ(CA+NC). Δ(MA+2/3 CA) contains a deletion of gag coding sequences including the N-terminal two-thirds of CA and most of the MA (deletion from MA codon 17 to CA codon 150). Constructs Δ(MA+CA) and ΔGAG were derived from combinations of Δ(MA+2/3 CA) with ΔCA-4 or with Δ(CA+NC), respectively. Δ(NC+PR) has a deletion of sequences encoding the NC, p2, and the PR, and Δ(GAG+PR) was derived from a combination of Δ(NC+PR) and ΔGAG. (B) Viral DNA sequences and encoded protein sequences of mutated HIV regions. HIV nucleotide positions are indicated, and inserted or altered amino acid residues are shown in boldface.

FIG. 2.

Western immunoblotting analysis of HIV proteins expressed and released from cells coexpressing Pr55^gag and Pr160^gag-pol. 293T cells were transfected or cotransfected with the designated plasmids. Fifteen micrograms of each plasmid was used for individual transfections, and 10 μg of pGAG and 2 μg for each of the GPfs constructs were used for cotransfection. At 48 h posttransfection, cells and supernatants were collected and prepared for protein analysis as described in Materials and Methods. Cell samples corresponding to 4% of the total samples and supernatant samples corresponding to 50% of the total samples were fractionated by SDS-10% PAGE and electroblotted onto a nitrocellulose filter. HIV Gag proteins were detected with an anti-p24^gag monoclonal antibody (B) or together with an additional anti-p17^gag monoclonal antibody (A, C, and D) at 1:5,000 dilution, followed by a secondary alkaline phosphatase-conjugated horse anti-mouse antibody at 1:5,000 dilution, and alkaline phosphatase activity was determined. The positions of molecular size markers (Std.) and those of HIV Gag proteins Pr55, p41, p24, and p17 are indicated. The arrow in panel A indicates the p24-associated Gag product produced by both ΔNC and Δ(MA+NC) that migrated slower than the p24^gag.

FIG. 2.

Western immunoblotting analysis of HIV proteins expressed and released from cells coexpressing Pr55^gag and Pr160^gag-pol. 293T cells were transfected or cotransfected with the designated plasmids. Fifteen micrograms of each plasmid was used for individual transfections, and 10 μg of pGAG and 2 μg for each of the GPfs constructs were used for cotransfection. At 48 h posttransfection, cells and supernatants were collected and prepared for protein analysis as described in Materials and Methods. Cell samples corresponding to 4% of the total samples and supernatant samples corresponding to 50% of the total samples were fractionated by SDS-10% PAGE and electroblotted onto a nitrocellulose filter. HIV Gag proteins were detected with an anti-p24^gag monoclonal antibody (B) or together with an additional anti-p17^gag monoclonal antibody (A, C, and D) at 1:5,000 dilution, followed by a secondary alkaline phosphatase-conjugated horse anti-mouse antibody at 1:5,000 dilution, and alkaline phosphatase activity was determined. The positions of molecular size markers (Std.) and those of HIV Gag proteins Pr55, p41, p24, and p17 are indicated. The arrow in panel A indicates the p24-associated Gag product produced by both ΔNC and Δ(MA+NC) that migrated slower than the p24^gag.

FIG. 3.

Pr160^gag-pol lacking most of the gag coding sequence could be incorporated into virus particles. (A) 293T cells were cotransfected with 10 μg of pGAG plus the indicated amount of GPfs or ΔGAG plasmid DNA. The total DNA in each transfection mixture was kept constant by addition of pBlueScript SK. At 48 h posttransfection, culture supernatants and cells were collected, prepared, and resolved on SDS-10% (A) or SDS-8% (B) PAGE gels. Viral proteins were probed with an anti-p24^gag monoclonal antibody (A) or an HIV-positive human serum (B). The positions of molecular size markers (Std.) and those of HIV Gag-Pol precursor and Gag proteins Pr55, p41, and p24 are indicated.

FIG. 4.

Expression and incorporation of protease-defective (PR⁻) Pr160^gag-pol deletion mutants into Pr55^gag particles. (A and B) 293T cells were transfected with the plasmid indicated. The HIV PR in all GPfs constructs was either inactivated or truncated. For cotransfection with pGAG, 1 μg of each plasmid DNA and 10 μg of pGAG were used, with addition of 9 μg of pBlueScript SK to a total amount of 20 μg of plasmid DNA. Two days after transfection, cells and supernatants were collected for protein analysis as described in Materials and Methods. Samples were fractionated by SDS-8% PAGE and then subjected to Western immunoblot analysis. Membrane-bound HIV proteins were probed with an HIV-positive human serum at a dilution of 1:10,000, followed by a secondary goat antihuman HRP-conjugated antibody at 1:10,000 dilution. (C and D) Incorporation of nonmyristylated Gag-Pol into virus particles. Methodology and plasmids used for analysis were identical to those in panels A and B, except that the GPfs constructs were expressed on a nonmyristylated backbone. The positions of the molecular size markers (Std.) and those of the HIV Pr55^gag and Pr160^gag-pol are indicated.

FIG. 4.

Expression and incorporation of protease-defective (PR⁻) Pr160^gag-pol deletion mutants into Pr55^gag particles. (A and B) 293T cells were transfected with the plasmid indicated. The HIV PR in all GPfs constructs was either inactivated or truncated. For cotransfection with pGAG, 1 μg of each plasmid DNA and 10 μg of pGAG were used, with addition of 9 μg of pBlueScript SK to a total amount of 20 μg of plasmid DNA. Two days after transfection, cells and supernatants were collected for protein analysis as described in Materials and Methods. Samples were fractionated by SDS-8% PAGE and then subjected to Western immunoblot analysis. Membrane-bound HIV proteins were probed with an HIV-positive human serum at a dilution of 1:10,000, followed by a secondary goat antihuman HRP-conjugated antibody at 1:10,000 dilution. (C and D) Incorporation of nonmyristylated Gag-Pol into virus particles. Methodology and plasmids used for analysis were identical to those in panels A and B, except that the GPfs constructs were expressed on a nonmyristylated backbone. The positions of the molecular size markers (Std.) and those of the HIV Pr55^gag and Pr160^gag-pol are indicated.

FIG. 5.

Release of HIV RT activity from cells cotransfected with the Pr160^gag-pol deletion mutants and pGAG. 293T cells were cotransfected with 1 μg of each PR-defective GPfs construct and 10 μg of pGAG. At 48 h posttransfection, supernatants were collected, filtered, and pelleted through a 20% sucrose cushion as described in Materials and Methods. Viral pellets were suspended in TSE, and about 40% of the suspensions were subjected to Western immunoblot analysis. The remaining suspensions were further diluted by addition of TSE and were then aliquoted for in vitro RT assays. RT activities (open bars) in each experiment were normalized to that obtained with GPfs plus pGAG, which was set at 100. Relative levels of released RT activity for each nonmyristylated GPfs deletion mutant were determined (shaded bars) by dividing the mutant GPfs RT activity by that of wt GPfs in parallel experiments. Values for each construct are derived from at least three independent experiments. Error bars indicate standard deviations.