Cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding defect

Abstract

The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.

FIG. 1.

(A) Schematic representation of the HIV-1 Gag protein (strain NL4-3) and the position of lysine residues within Gag. (B) Detection of ubiquitinated MA, CA, NC, SP2, and p6 in mature virions. 293T cells were cotransfected with the indicated pΔR-derived plasmids and pHA-Ub (+) as indicated. Mutants are designated as described in the text. Forty-eight hours after transfection, virions were pelleted, and soluble contaminants were removed by trypsin digestion where indicated (+). Pellet fractions were normalized for CA content and analyzed by Western blotting with anti-HA antibody (upper panel). The blot was reprobed with anti-CA antiserum (lower panel). Mature ubiquitinated Gag proteins and HA-Ub are identified on the right. Molecular mass standards (in kilodaltons) are shown at the left side of each panel. An HA-reactive protein that was removed by trypsin treatment is marked by an asterisk.

FIG. 2.

Ubiquitination of cell-associated wt and mutant Gag proteins. Extracts from 293T cells coexpressing HA-Ub with wt and mutant pΔR/PR(−) constructs were prepared under denaturing conditions and immunoprecipitated for Gag. Equal amounts of Gag proteins were analyzed for Ub modification by Western blotting with an anti-HA antibody (upper panel). The blot was reprobed with anti-CA antiserum (lower panel). Please note that on our gel system wt Gag travels below 55 kDa, and p6(KR) mutant Gag protein consistently travels lower than wt Gag, which could account for the shift of the lower Gag-Ub conjugate in p6 mutant samples (e.g., lane 4).

FIG. 3.

Infectivity of Gag lysine mutants. 293T cells were transfected with wt or lysine mutant pNL4-3 proviral constructs. Virus-containing supernatants were harvested 24 h after transfection, cleared, and analyzed for virus content by ELISA (values in nanograms/milliliter are given at the bottom of the panel) and titrated on TZM indicator cells. The infectivity was normalized for CA content and is given in infectious units (i.u.)/nanogram CA.

FIG. 4.

(A) Levels of virus release by Gag lysine mutants. HeLaP4 cells were transfected with the indicated constructs and, 24 h after transfection, were pulse labeled with [³⁵S]methionine. After a 2.5-h chase period, cell (upper panel) and virus (lower panel) samples were immunoprecipitated with sheep anti-CA antiserum and visualized by autoradiography. Viral proteins are identified on the right. The band marked by an asterisk represents an unknown CA-reactive protein that is strongly diminished upon mutation of the NC lysine residues. (B) Quantification of wt and NCSP2p6(KR) release kinetics. A pulse-chase analysis of wt and NCSP2p6(KR) mutant virus release was carried out in triplicate and quantified. The signals from Gag, MACASP1, CASP1, and CA were quantified and normalized for the methionine content of each protein. The release efficiency was calculated as the signal from virus-associated CA proteins divided by the sum of the signals from cell- and virus-associated CA proteins.

FIG. 5.

Thin-section electron microscopy analysis of HeLaP4 cells transfected with wt (a), NC(KR) (b), NCSP2(KR) (c), or NCp6(KR) (d) mutant pNL4-3. Bars: 500 nm (a), 200 nm (b), 500 nm (c), and 200 nm (d).

FIG. 6.

Thin-section electron microscopy analysis of HeLaP4 cells transfected with NCSP2p6(KR) mutant pNL4-3. Bars: 500 nm (a to c) and 100 nm (d).

FIG. 7.

Quantification of thin-section electron microscopical data from HeLaP4 cells transfected with wt or Lys mutant pNL4-3 constructs. (A) Percentages of released wt and mutant virions and budding structures. In all cases except for SP2p6(KR), three different transfections and four epon blocks were analyzed. In the case of SP2p6(KR), one epon block each from two transfections was analyzed. For this analysis, we distinguished between “free” virions (which could be immature or mature and for which no connection to the cell membrane was visible; gray bars) and budding structures for which a visible membrane connection to the cell membrane existed (black bars). Numbers of virus structures counted are the following: 345 (wt), 428 [NC(KR)], 470 [NCSP2(KR)], 426 [NCp6(KR)], 180 [SP2p6(KR)], and 465 [NCSP2p6(KR)]. Percentages of free virions (gray bars) were predicted using linear regression. The Pearson product-moment correlation coefficients were the following: 0.99 (wt), 0.97 [NC(KR)], 0.8 [NCSP2(KR)], 0.85 [NCp6(KR)], and 0.98 NCSP2p6(KR)]. Percentages of budding structures were calculated as 100 minus the predicted percentage of free virions. (B) Quantification of virus budding structures per micrometer plasma membrane length. The total numbers of virus structures observed were 90 (wt, of which 16 were budding structures) and 181 {[NCSP2p6(KR)], of which 91 were budding structures}. The total lengths of plasma membrane analyzed were 567 mm (wt) and 440 μm [NCSP2p6(KR)]. Error bars represent the standard errors of the means.