Effects of reduced gag cleavage efficiency on HIV-1 Gag-Pol package

Abstract

Background: HIV-1 pol, which encodes enzymes required for virus replication, is initially translated as a Gag-Pol fusion protein. Gag-Pol is incorporated into virions via interactions with Gag precursor Pr55^gag. Protease (PR) embedded in Gag-Pol mediates the proteolytic processing of both Pr55gag and Gag-Pol during or soon after virus particle release from cells. Since efficient Gag-Pol viral incorporation depends on interaction with Pr55^gag via its N-terminal Gag domain, the prevention of premature Gag cleavage may alleviate Gag-Pol packaging deficiencies associated with cleavage enhancement from PR.

Results: We engineered PR cleavage-blocking Gag mutations with the potential to significantly reduce Gag processing efficiency. Such mutations may mitigate the negative effects of enhanced PR activation on virus assembly and Gag-Pol packaging due to an RT dimerization enhancer or leucine zipper dimerization motif. When co-expressed with Pr55^gag, we noted that enhanced PR activation resulted in reduced Gag-Pol cis or trans incorporation into Pr55^gag particles, regardless of whether or not Gag cleavage sites within Gag-Pol were blocked.

Conclusions: Our data suggest that the amount of HIV-1 Gag-Pol or Pol viral incorporation is largely dependent on virus particle production, and that cleavage blocking in the Gag-Pol N-terminal Gag domain does not exert significant impacts on Pol packaging.

Keywords: Gag; Gag cleavage; Gag-Pol; HIV-1; Protease; Virus assembly.

Fig. 1

Effects of Gag cleavage site mutations on virus assembly and processing. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. Indicated are the HIV-1 Gag protein domains MA (matrix), CA (capsid), p2, NC (nucleocapsid), p1 and p6, and pol-encoded p6*, PR, RT and IN. Arrowheads indicate where alanines (A/A) were substituted for amino acid residues at the MA/CA (Y/P) and p2/NC (M/M) junctions. b-d Enhanced PR activation by efavirenz (EFV) reduced virus yields and Gag-Pol packaging. 293T cells were transfected with designated constructs. At 18 h post-transfection, transfectants were equally split, placed on two plates, and either treated or mock-treated with 1 µM EFV. At 4 h post-treatment, culture supernatants were removed and replaced with medium containing EFV with or without an HIV-1 protease inhibitor (panel d). Shown is a representative immunoblot from two independent experiments. Cells and supernatants were harvested for Western immunoblot analysis 48 h after medium replacement. HIV-1 Gag proteins were probed with anti-p24CA and anti-p17MA monoclonal antibodies. Anti-RT serum was used to detect RT. Indicated are positions of the RT p66 and p51 subunits, Pr55^gag, p4^1gag, p24^gag and p17^gag

Fig. 2

Effects of Gag cleavage mutation on Gag-Pol trans incorporation into virus particles. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. Gag domains, pol-encoded products, and Gag cleavage site mutations are identical to those described in the Fig. 1 caption. GPfs and CSMfs both contained the same gag/pol frameshifting mutation to force Gag-Pol expression without Gag. b 293T cells were transfected with indicated amounts of GPfs or CSMfs plasmid DNA, either alone or with 10 µg Gag plasmid DNA at a ratio of 1:10 (lanes 2 and 5) or 1:1 (lanes 3 and 6). c 293T cells were transfected with 10 µg GPfs or CSMfs plasmid DNA, either alone or with 10 µg Gag (lanes 5–6) or CSMGag (lanes 8–9) plasmid DNA. Ten µg of pBluescript (SK) plasmid DNA was added to give a total of 20 µg DNA for each transfection. d 293T cells were co-transfected with either Gag (10 µg) or CSMGag (10 µg) plus either GPfs (1 µg) or CSMfs (1 µg) expression vectors. At 18 h post-transfection, transfectants were equally split, placed on two plates, and either treated or mock-treated with 1 µM efavirenz (EFV). After an additional 4 h (22 h post-transfection), culture supernatants were removed and replaced with medium containing EFV. Cells and supernatants were harvested for Western immunoblot analysis 48 h post-medium replacement. b-d Show is a representative immunoblot from three similar independent experiments

Fig. 3

Incorporation of PR-defective Gag-Pol into wt virus particles. a Schematic representations of HIV-1 Gag and Gag-Pol expression vectors. Indicated are the HIV-1 Gag protein domains MA (matrix), CA (capsid), p2, NC (nucleocapsid), p1 and p6, and pol-encoded p6*, PR, RT and IN, all as described in the Fig. 1 caption. “X” denotes a substitution mutation at the PR catalytic Asp residue. The C-termini of constructs fsDmyc and CSMfsDmyc are attached with myc epitopes. b-e 293T cells were transfected with 10 µg of wt plasmid DNA plus the indicated amounts of fsDmyc or CSMfsDmyc plasmid DNA. At 48 h post-transfection, cells and supernatants were collected, prepared, and subjected to Western immunoblotting. Gag proteins were probed with anti-p24CA and anti-p17MA monoclonal antibodies. Panel c was derived from panel b following sequential probing with anti-RT, anti-Myc or anti-IN antibodies. Shown is a representative from three independent experiments

Fig. 4

Virus yields and Gag-Pol packaging reduced by enhanced Gag-Pol autocleavage. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. “X” denotes a PR-inactivated mutation. p6* adjacent to the active PR domain was replaced with a leucine zipper (LZ) as denoted by the striped (Wz) box. Remaining C-terminal p6* residues are in boldface. Arrows denote PR cleavage sites. Altered or additional residues are in italics. b 293T cells were transfected with designated constructs. Two days post-transfection, cells and supernatants were collected, prepared, and subjected to Western immunoblotting. c Relative levels of virus-associated RT. Levels of HIV-1 p24gag-associated Gag proteins Pr55, p41, and p24 and RT-associated Pol proteins Gag-Pol, Pol and p66/51RT were quantified by scanning immunoblot band densities. Ratios of total virus-associated RT versus Gag protein levels were calculated for each sample and normalized to the wt in parallel experiments. All data were obtained from three independent experiments. Error bars indicate standard deviation *p < 0.05

Fig. 5

Enhanced Gag-Pol autocleavage reduced Gag-Pol trans incorporation into Gag particles. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. Designated constructs are identical to those shown in Fig. 4a, but expressed in a gag/pol frameshift mutation backbone (GPfs). b-c 293T cells were transfect with WT or W402A or co-transfected with the designated construct plus a Pr55gag (Gag) expression vector at a plasmid DNA ratio of 1:10. SK denotes the plasmid pBluescript. At 48–72 h post-transfection, cells and supernatants were collected, prepared, and subjected to Western immunoblotting. Relative levels of virus-associated RT are indicated at bottom. d Relative levels of virus-associated RT were determined as described in the Fig. 4 caption. All data are from three independent experiments. Error bars indicate standard deviation *p < 0.05