The Y132I mutation displays a strong transdominant effect on wild-type viral infectivity in a phenotypic mixing experiment. (A) Relative potencies of Y132I, L363I, M377I, N432I, F448I, and D25A-PR mutants. Mutant and wild-type plasmid DNAs were cotransfected into 293T cells, gradually increasing the percentage of mutant from 0% to 100%. The amounts of mutant used were 0%, 10%, 20%, 40%, 60%, 80%, 90%, and 100% in each cotransfection experiment except for the Y132I mutant, where 5% mutant was added. Viral infectivity was considered to be 100% when 0% mutant was used (i.e., 100% wild type), and all other viral infectivities obtained from different mutant fractions were compared to the 100% value for the wild type. Unless specified, all the infectivity data are shown as means ± standard errors. (B) Relative viral infectivity of the Y132I mutant at a low range of mutant percentages, from 0% to 20%. (C) Western analysis of virion particles produced from transfection with wild-type and mutant DNAs. Lane 1 represents a mock transfection where no DNA was used. The percentage of mutant used for each cotransfection is shown. Open arrowheads indicate processed CA protein, and closed arrowheads in the Y132I, L363I, M377I, and D25A-PR mutants indicate the MA/CA fusion protein, CA/SP1 fusion protein, CA/SP1/NC fusion protein, and unprocessed Gag precursor, respectively. (D) Western analysis showing processed RT in viral particles of the Y132I and Y132I(W80/M20) viruses probed with an anti-HIV-1 RT antibody. Closed arrowheads indicate the completely processed p66 and p51 subunits of RT. (E) Distribution of virion morphology. Total numbers of viruses counted were 96 for the wild type (WT), 147 for the Y132I mutant, and 72 for the Y132I(W80/M20) mutant. The fractions of mature, immature, eccentric, and aberrant viruses are shown. (F) Morphology of Y132I viruses and viruses from cotransfection with a ratio of 80% wild type and 20% Y132I [Y132I(W80/M20)] assessed by thin-section EM. Examples of wild-type viruses are shown in the first row. The phenotype of each virion is labeled. The bars in each image represent 100 nm.

Mutations blocking salt bridge formation do not show potency equivalent to that of the Y132I mutant. (A) Relative potency of CA D51A, P1L, and P1F mutations. Phenotypic mixing experiments were performed according to the protocol described in the legend of Fig. 2. (B) Western analysis of viral particles harvested from the supernatant of 293T cells transfected with mutant and wild-type DNAs. The percentage of the mutant used for each cotransfection is shown. Open arrowheads indicate processed CA proteins.

Ile substitutions at the P1 position of Gag cleavage sites affect Gag processing and impair viral infectivity. (A) Schematic diagram showing protein domains of the HIV-1 Gag protein. Amino acid sequences of the P1 position of the cleavage sites are shown in boldface type. The Ile substitution mutants are shown with the cleavage site. Arrows at the top indicate five different cleavage sites in Gag. (B) Relative infectivity of the P1 Ile mutant viruses. Culture supernatants of 293T cells were harvested 48 h after transfection and used to infect TZM-BL reporter cells. The TZM-BL cells were lysed and used in a luciferase assay 48 h after infection to assess the level of infection. The measurements of infectivity from the luciferase assays were normalized by the amount of viral genomic RNA in the infecting virus quantified by real-time PCR analysis. Measurements from the infectious molecular clone pNL-CH (wild type [WT]; derived from pNL4-3) were used as the wild type and were considered to be 100%. The mean data with standard errors from several experiments are shown. All the infection data shown in this paper were obtained by following the same protocol unless otherwise specified. (C) Western analysis of viral particles. Culture supernatants harvested 48 h after transfection were subjected to ultracentrifugation to concentrate the viral particles. The pelleted viral particles were analyzed by Western analysis using either anti-CA antibody (top) or anti-NC antibody (bottom) as the primary antibody.

Inhibition of myristoylation of the Gag protein does not relieve viral infectivity of the viruses containing either 5% or 10% of the Y132I mutant. (A) Relative potency of a mutant lacking myristoylation (G2A). (B) Relative infectivity of the virus containing either 5% or 10% of the Y132I mutant in the presence of the myristoylation inhibitor 2-hydroxymyristic acid. 293T cells were transfected with wild-type and mutant DNAs, and the drug was added into the medium 4 h after transfection. Culture supernatants were harvested 48 h after transfection, and infectivity data were obtained according to the protocol described in the legend of Fig. 2. Mean data are shown.

Viral DNA synthesis in U87.CD4.CXCR4 cells infected with wild-type (WT) and Y132I and Y132I(W80/M20) mutant viruses. A 293T cell culture supernatant harvested at 48 h after transfection was used to infect U87.CD4.CXCR4 cells. Total DNA was isolated from the infected cells harvested at 24 h postinfection. To detect early viral DNA products, primers derived from the env gene were used. A separate set of primers was used to detect HIV-1 2-LTR circles as evidence of the synthesis of full-length viral DNA and import into the nucleus. For comparison, the PCR amplification of the wild-type sample was done in a dilution (dil.) series. Mock is a DNA sample isolated from cells infected with the culture supernatant from mock transfection (no DNA). In other experiments, we have shown that the PCR signal generated using this protocol is abolished if a viral DNA synthesis inhibitor is included at the time of infection. In this experiment, the lack of signal from the infection with the Y132I virus is consistent with a lack of transfected plasmid DNA being carried over to the infection.