Infectivities of recombinant viruses with HIV-1 capsid changes. Cell supernatants containing recombinant wt HIV-GFP or HIV-GFP with the indicated capsid changes were assessed for RT activity. Then 5 × 10⁴ RT units of virus was added to human, MK2D, and Vero cells, and 1 × 10⁵ RT units of virus was added to PRL cells. Cells were incubated for 3 days prior to FACS analysis for GFP expression. The average percentages of GFP-expressing cells and the standard deviations were calculated from at least two independent experiments.

Effect of CspA treatment of target cells on infectivities of wt and mutant viruses. Target cells were treated with CspA (1 μg/ml) or the equivalent amount of dimethyl sulfoxide solvent 30 min prior to the addition of virus. Cell supernatants containing wt HIV-GFP (wt HIV-1) or the indicated mutant HIV-GFP viruses were normalized for RT activity and added to HeLa cells at 3 × 10⁴ RT units/well or to PRL and OMK cells at 1 × 10⁵ RT units/well. Cells were incubated for 3 days prior to FACS analysis for GFP expression. The means and standard deviations from duplicate experiments are shown.

Combined effects of competitor VLPs and CspA treatment on wt HIV-1 infectivity in PRL cells. Cell supernatants containing the indicated wt and mutant HIV-1 VLPs were added to PRL cells at 2 × 10⁵ RT units/well following treatment of the cells for 30 min with either CspA at 1 μg/ml or control dimethyl sulfoxide solvent. Control cells were not incubated with VLPs (none) or were incubated with VLPs lacking envelope glycoproteins [HIV-1(env⁻)]. The HIV-GFP virus was immediately added to the cells at 4 × 10⁴ RT units/well. Cells were incubated for 3 days before being harvested for FACS analysis. The values represent the means and standard deviations derived from two independent experiments.

Competition for target cell restriction factors with wt and mutant HIV-1 VLPs. (A) The indicated amounts, in RT units, of VLPs were added to HeLa and PRL cells. Immediately thereafter, recombinant wt HIV-GFP virus was added to HeLa cells at 1 × 10⁴ RT units/well or to PRL cells at 4 × 10⁴ RT units/well. Samples were incubated for 3 days and analyzed by FACS. Labels indicate the identity of each VLP used. All VLPs were pseudotyped with VSV-G, except for the control, a wt HIV-1 VLP with no envelope glycoproteins [HIV-1(env⁻)]. (B) Approximately 1.5 × 10⁵ RT units of the indicated VLPs/well were added to HeLa, PRL, and OMK cells. Immediately thereafter, the HIV-GFP virus was added to HeLa cells at 1 × 10⁴ RT units/well or to PRL and OMK cells at 4 × 10⁴ RT units/well. The percentages of cells that were positive for GFP expression were determined by FACS. The identities of the VLPs used for competition are indicated at the bottom of the figure.

Infectivity of viruses in primary rhesus monkey cells. Mutant and wt reporter virus cell supernatants containing wt HIV-GFP and HIV-GFP with the indicated mutant capsids were normalized for RT activity and added at a concentration of 10⁵ RT units/well to target cells. The target cells consisted of primary rhesus macaque macrophages purified from whole blood (A) or PRL fibroblasts (B). Cells were incubated with recombinant HIV-1 vectors for 3 days and then analyzed by microscopy and FACS. Fluorescence microscopy was performed by using a fluorescein isothiocyanate filter set on a Nikon TE300 inverted microscope. The results shown are representative of those obtained from two independent experiments.

Infection of cells by recombinant viruses with changes in HIV-1 capsid. Cell supernatants containing the indicated GFP-expressing viruses were assayed for RT activity. Viruses were added to cells at 1.6 × 10⁴ RT units/well or threefold serial dilutions thereof. Samples were incubated for 3 days and assayed for GFP expression by FACS analysis. The mean percentages of GFP-positive cells and standard deviations were calculated from two independent experiments. The cell lines used originated from the following species: HeLa, human; Vero, African green monkey; PRL, rhesus macaque; MK2D, rhesus macaque; OMK, owl monkey; and Pindak, squirrel monkey. The control wt HIV-1 and HIV(SCA) viruses are indicated by solid lines, and the mutant viruses are indicated by broken lines.

Wild-type and mutant virion proteins. 293T cells were transfected with plasmids encoding the wt and mutant HIV-1 Gag/Pol proteins. Except for the control in lane 16, cells were cotransfected with a vector encoding CypA-HA. Metabolically labeled supernatants were pelleted through a 20% sucrose cushion and resuspended in radioimmunoprecipitation assay buffer. (A) [³⁵S]Methionine-labeled viral proteins were separated by SDS-12.5% PAGE and visualized by autoradiography. The locations of the p41 (MA plus CA) and CA proteins as well as CypA are shown on the left. Values on the right represent the molecular masses in kilodaltons of the marker proteins (far right lane). (B) Solubilized virion pellets were separated by SDS-4 to 12% PAGE, transferred to nitrocellulose membranes, and blotted with a high-affinity rat anti-HA antibody (3F10). The viral samples in panels A and B were identical.

HIV-1 CA sequences. (A) The amino acid sequences of HIV-1_HXBc2 (top) and SIV_mac (bottom) CA proteins were aligned. Vertical lines indicate sequence identity and dots represent related residues. Differences between the two proteins in solvent-accessible residues were determined through the analysis of GRASP computer models of the molecular surface of the HIV-1 CA protein and are colored in the HIV-1 CA sequence (top). Residues that were changed in this study from the HIV-1 sequence to the SIV_mac sequence are shown in red in the HIV-1 CA sequence. Horizontal lines above the HIV-1 sequence designate structurally indeterminate regions. (B) Structural model of the HIV-1 CA protein (1). The N- and C-terminal domains are designated NTD and CTD, respectively. The hexamer symmetry axis is indicated, with the arrow pointing towards the exterior of the viral core (34). With respect to the figure on the left, the right-hand figure was rotated 50° about the x axis and −10° about the y axis. From this perspective, the ridge formed at the interface of helices 3 (magenta) and 6 (orange) is evident. Also highlighted are the CypA-binding loop (cyan) and the interdomain linker (green). (C) Structure of the HIV-1 CA protein in the same orientation as that of the model at the right side of panel B. The three regions in which changes result in escape from postentry restrictions in monkey cells are highlighted as follows: the helix 3/6 ridge is red, the CypA-binding loop is blue, and the interdomain linker is magenta. The side chains of residues associated with the indicated changes are shown.