Gag Molecules Research Articles

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.

Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events.

The gag precursor polyprotein of Moloney murine leukemia virus (MuLV) is normally modified by myristylation of the N-terminal glycine. Previous work showed that the Pr65gag lacking the myristylation site does not associate with cellular membranes or assemble into virus particles. We now report that it also is not cleaved to the mature gag cleavage products within the cell and that it sediments as a free 65-kilodalton monomer in detergent-free cell extracts containing 0.3 M NaCl. Even when the cells containing the mutant are productively infected with wild-type MuLV, the mutant Pr65gag is not processed into cleavage products and is not incorporated into the virions produced by these cells. Thus, the mutant gag molecules seem unable to participate in the normal processes of self-assembly and maturation. We propose that myristate-mediated membrane association is an essential first step in MuLV assembly. This association may also play a role in budding of MuLV.

4520412 Autostop mechanism for tape recorder: Kobun Yoshida, Saitama, Japan assigned to Pioneer Electronic Corporation

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.

Cytoskeleton-associated Pr65 gag and retrovirus assembly

Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65 gag with cytoskeletal elements in infected mouse fibroblasts. The Pr65 gag associated with Nonidet P-40 (NP-40)-insoluble cytoskeletal structures appears to be subphosphorylated in comparison to NP-40-soluble Pr65 gag The association of Pr65 gag with skeletal elements can be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent, or with buffers of high ionic strength. Both the skeleton-associated Pr65 gag and its NP-40-soluble counterpart can be labeled with [ 3H]palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65 gag molecules bound to skeletal elements in the infected cell appear to be more stable to proteolytic processing than NP-40-soluble Pr65 gag While the association of Pr65 gag with cytoskeleton elements in the cell is neither increased nor decreased by blocking virus assembly and release with interferon, Pr65 gag appears to accumulate in the cytoskeleton-enriched fraction of cells chronically infected with a temperature sensitive mutant of R-MuLV ( ts 17) when such cells are grown at the nonpermissive temperature. Based on these and other results, we have proposed a model for the active role of cytoskeleton associated Pr65 gag in retrovirus assembly.

Comparative immunofluorescence of murine leukemia virus-derived membrane-associated antigens

We recently reported that the distribution and location of Moloney murine leukemia virus-derived membrane-associated gp70 and p30 antigens on the surface of 3.7% formaldehyde-fixed, chronically infected mouse fibroblasts were completely distinct, as judged by immunofluorescent light microscopy ( M. Satake, P. N. McMillan, and R. B. Luftig (1981). gp70, one of the two env gene products, exhibited a multiple-dot fluorescent pattern on the external surface of infected cells, while p30, one of the gag gene products, exhibited a diffuse fluorescence pattern which was apparently derived from Pr65 gag molecules associated with the cytoplasmic face of the cell membrane. We have now examined the membrane fluorescence patterns of p15E, the other env gene product, as well as p15, p12, and p10, the other gag gene products. In these studies, both multivalent and monoclonal antibodies as well as fluorescenn- and rhodamine-conjugated probes were used. We found that: (i) each of the env gene products, gp70 and p15E, exhibited characteristic and distinctive multiple-dot staining patterns. Further, each protein was labeled on intact cells with 125I-protein A plus homologous antiserum, confirming that both gp70 and p15E had externally exposed antigenic determinants. (ii) Among the gag gene products, p15 exhibited a different membrane fluorescence pattern than the diffuse pattern seen with p30, p12, and p10. The p15 pattern had an additional multiple-dot component. (iii) By double immunofluorescence we observed that the p15E and p15 multiple-dot patterns were superimposable at the same loci on infected cells. These three results suggest, first, that the cleavage of gp70 and p15E occurs prior to the arrival of the env polyprotein precursor at the cell surface and, second, there is an association between p15E and p15 antigenic determinants at the cell membrane. This latter association between an env and a gag gene product may be important for viral assembly.

Gene order of mouse mammary tumor virus precursor polyproteins and their interaction leading to the formation of a virus

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75 env containing gp52 and gp36, and Pr75 gag containing p27, pp20, p14, and p10. The gene order for gPr75 env has been previously shown to be H 2N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75 gag is H 2N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [ 51Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of 125I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only 125I-gp52. In contrast, cell surface glycoprotein labeling revealed [ 3H]gp52 and [ 3H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75 gag and gp52 but not gPr75 env . Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75 gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.