Human immunodeficiency virus type 1 Vpr protein is incorporated into the virion in significantly smaller amounts than gag and is phosphorylated in infected cells

Abstract

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is a small accessory protein involved in the nuclear import of viral DNA and the growth arrest of host cells. Several studies have demonstrated that a significant amount of Vpr is incorporated into the virus particle via interaction with the p6 domain of Gag, and it is generally assumed that Vpr is packaged in equimolar ratio to Gag. We have quantitated the relative amount of Vpr in purified virions following [(35)S]cysteine labeling of infected MT-4 cells, as well as by quantitative immunoblotting and found that Vpr is present in a molar ratio of approximately 1:7 compared to capsid. Analysis of isolated core particles showed that Vpr is associated with the mature viral core, despite quantitative loss of p6 from core preparations. Metabolic labeling of infected cells with ortho[(32)P]phosphate revealed that a small fraction of Vpr is phosphorylated in virions and infected cells.

FIG. 1

Identification of [³⁵S]cysteine-labeled virion proteins by immunoprecipitation. A total of 3 × 10⁵ infected cells (A) or purified virus equivalent to 2.4 ml of tissue culture supernatant (B) were lysed in 750 μl of radioimmunoprecipitation assay buffer containing 2 mM Pefabloc, 10 μM E64, and 1 μM pepstatin. Extracts were subjected to immunoprecipitation according to standard procedures (16), using polyclonal rabbit antisera directed against the indicated HIV proteins or an unrelated antibody (control) bound to protein A-agarose (Roche). Immunoprecipitates were separated by SDS-PAGE (17.5% acrylamide; acrylamide/bisacrylamide, 200:1) and visualized by phosphorimage analysis using a Fuji BAS2000 instrument. As a reference, an aliquot of purified labeled virus was also loaded directly onto the gel (lane ³⁵S virus). Positions of marker proteins and their molecular masses in kilodaltons are indicated at the left.

FIG. 2

Determination of the relative amounts of Vpr and CA in virus lysates. (A) Aliquot of a purified ³⁵S-labeled virus sample used for quantitative analysis. The sample was analyzed by SDS-PAGE followed by silver staining as described by Heukeshoven and Dernick (19) (lane 1) as well as phosphorimage detection (lane 2). (B) Radioactivity in bands corresponding to Vpr, MA, CA, and IN contained in virus samples as determined by phosphorimage analysis. After normalizing for the number of cysteines present, average numbers of molecules were calculated relative to the intensity of the CA band in the same lane, which was arbitrarily set at 100%. Data shown represent the mean relative value calculated for each protein. (C) Relative amounts of virion-associated CA and Vpr as determined by immunoblotting. Various amounts of purified virus were separated by SDS-PAGE in parallel to serial dilutions of recombinant CA protein or synthetic Vpr protein of known concentration. Proteins were detected by immunoblotting using rabbit antisera directed against CA or Vpr followed by enhanced chemiluminescence staining. Band intensity was measured by densitometry using a Desaga CD50 instrument.

FIG. 3

Immunoblot analysis of purified HIV-1 virions and isolated core particles. Mature core particles were prepared as described by Welker et al. (42). Virus particles pelleted through a sucrose cushion (lane 1) and further purified by banding in an OptiPrep gradient (lane 2) as well as a preparation of core particles from OptiPrep gradient-purified virions (lane 3) were separated by SDS-PAGE. Similar amounts with respect to CA were loaded in each lane. Virion-associated proteins were identified by immunoblotting using a mixture of antisera against the indicated proteins and enhanced chemiluminescence staining. CypA, cyclophilin A.

FIG. 4

Immunoprecipitation of ³²P-labeled HIV-1 proteins. Lysate from 3.5 × 10⁶ infected cells metabolically labeled with ortho[³²P]phosphate (A) or purified virus equivalent to 18 ml (Vpr) or 2.25 ml (MA and CA) of tissue culture supernatant (B) was subjected to immunoprecipitation as in Fig. 1, using the indicated antisera. Immunoprecipitates were separated by SDS-PAGE and visualized by phosphorimage analysis. As a reference, an aliquot of [³⁵S]cysteine-labeled virus lysate was separated on the same gel (lane ³⁵S virus). Positions of marker proteins and their molecular masses in kilodaltons are indicated at the left.

FIG. 5

Analysis of virion-associated Vpr by two-dimensional gel electrophoresis. (A) Unlabeled virus released from infected MT-4 cells was gradient purified as described for the labeled virus preparations. A sample corresponding to 15 μg of CA was applied to an Immobiline DryStrip 3-10L (Amersham Pharmacia) and subjected to isoelectric focusing (IEF) under denaturing conditions (8 M urea) according to the manufacturer's instructions, using an IGphor unit. Separation in the second dimension was performed by SDS-PAGE; subsequently, Vpr was detected by immunoblotting using antiserum directed against synthetic Vpr. (B) Gradient-purified ³²P-labeled virus equivalent to 20 ml of tissue culture supernatant was separated by IEF (Immobiline DryStrip 6-11L) followed by SDS-PAGE. Radiolabeled protein was detected by phosphorimage analysis. Arrows indicate a single phosphorylated protein spot of the apparent molecular weight of Vpr, corresponding to an IEP of 6.3 (B) and an immunoreactive spot corresponding to the same IEP (A).