Palmitoylation of the Rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity

Abstract

The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed.

FIG. 1

Locations of mutations within RSV PrC gp37 (TM). The previously suggested boundaries of the MSD are indicated by the shaded box. Oligonucleotide mutagenesis was used to change the TGC codon for cysteine to a GGC codon for glycine to investigate the potential thioester linkage of palmitate at these sites. Mutants were named C164G, C167G, and C164G/C167G to denote the location and type of substitution at the cysteine codon in each mutant.

FIG. 2

Labeling of RSV glycoproteins with [³ H]palmitic acid. Wild-type and mutant glycoproteins expressed in CV-1 cells after infection with the recombinant SV40 vector were labeled for 4 h with [³H]palmitic acid. Immunoprecipitates were separated on SDS–12% PAGE and then analyzed by fluorography. Lane 1, wild type (wt); lane 2, C164G; lane 3, C167G; lane 4, C164G/C167G; lane 5, mock infected.

FIG. 3

Kinetics of glycoprotein processing and stability in the presence and absence of chloroquine. (A) Wild-type and mutant viral glycoproteins expressed from the recombinant SV40 expression vector system were pulse-labeled with [³H]leucine for 15 min (lanes P) and then chased for 2, 4, or 6 h (lanes 2, 4, and 6, respectively). (B) The wild-type and mutant glycoproteins were pulse-labeled and chased as for panel A, but the cells were preincubated in 100 μM chloroquine for 1 h prior to the pulse and chloroquine was present in the medium throughout the chase periods. Lane designations are as in panel A.

FIG. 4

Indirect immunofluorescence of wild-type (wt) and palmitoylation mutant glycoproteins in CV-1 cells reveals different steady-state distributions. The intracellular distributions of wild-type and mutant env gene products were probed after acetone fixation and permeabilization of recombinant-SV40-infected CV-1 cells grown on glass coverslips using rb-anti-RSV-TM-Cpep (Whole Cell IF). Steady-state surface expression of wild-type and mutant env gene products was detected after incubation of SV40 vector-infected unfixed CV-1 cells on ice with neutralizing chi-anti-RSV PrC antibody followed by ethanol-acetic acid fixation and secondary antibody incubation (Surface IF). Internalization of wild-type and mutant Env proteins from the plasma membrane into prelysosomal vesicles was visualized by incubating Env-expressing cells with chi-anti-RSV PrC antibody in the presence of chloroquine at 37°C for 2 h; the cells were then fixed and stained as for surface IF (Antibody Uptake). The brightly stained vesicles represent endocytic prelysosomal vesicles, most likely late endosomes (29, 39).

FIG. 5

Infectivity of wild-type (Wt) and mutant RSV genomes in avian cells. Primary TEC were transfected in duplicate with the respective pATV8R proviral DNA or mock transfected and passaged over 24 days. Supernatants were harvested at 4-day intervals. Virus spread was assayed by the RT assay as described in Materials and Methods. Mean RT values from a representative experiment are plotted. mock, mock-infected control.

FIG. 6

Sequence analysis of RT-PCR products from mutant and wild-type virions at day 24. Cells were transfected and cultured as for Fig. 5. Culture medium was harvested on day 24 posttransfection, virions were pelleted, and the extracted RNA was subjected to RT-PCR amplification. The resulting product was excised from a gel and sequenced directly. Wild-type T residues are denoted by the arrowheads, and mutant G residues are denoted by the circles.

FIG. 7

Incorporation of wild-type and mutant glycoproteins into virions released from avian cells. The stable chicken fibroblast cell line Df-1 was transfected with wild-type (wt) and mutant pATV8R DNAs or mock transfected; 44 h posttransfection, the cells were pulse-labeled with [³⁵S]methionine and chased for 6 h in complete medium. Virions were pelleted from the medium as described in Materials and Methods. Lysates of viral pellets and of cells were subjected to consecutive immunoprecipitation with anti-Env and anti-Gag sera. Viral proteins from cell lysates were separated on SDS–10% PAGE gels (A), as was virion-associated Env (B). Virus-associated Gag was visualized on an SDS–15% PAGE gel (B). (C) Amounts of p27 (CA), SU, and TM were quantitated, and relative ratios, adjusted to wild type, were plotted.

FIG. 8

Revised schematic organization of the RSV gp37 (TM) MSD. Palmitoyl groups are shown inserted into the hydrophobic environment of the bilayer, while the amine groups of the histidine and glutamine residues are located in the polar head group region. The long positively charged side chain of the arginine allows this region of the peptide backbone to be buried in the hydrophobic region of the lipid bilayer. The leucine (residue 165) that in mutant μ26 (9) is converted to an arginine is in this model located close to the inner membrane-cytoplasm boundary. This would allow insertion of the charged residue without significantly interfering with the membrane anchor properties of the region.