Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes

Abstract

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4 ؉ T lymphocytes, were evaluated by using a newly developed system. In CD4 ؉ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4 ؉ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4 ؉ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4 ؉ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.

Nucleocapsid and Matrix Protein Contributions to Selective Human Immunodeficiency Virus Type 1 Genomic RNA Packaging

“…Immature and mature particles were present among the cells transfected with the plasmid producing wild-type virus (pHXB2gpt), consistent with ongoing virus production 48 h after transfection. Immature and mature particles were also present among the cells transfected with plasmids pHX⌬MA, confirming previous observations (15,30,31), and pHX/MT-NC ( Fig. 3B), although in the case of virus from pHX/MT-NC, the mature particles were not as frequent as with the wild-type virus.…”

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

“…The wild-type infectious proviral plasmid HXB2 Hygro (Myrϩ) was constructed by replacing the neomycin resistance gene in the infectious proviral plasmid HXB2Neo (14) with the hygromycin resistance gene. The hygromycin resistance gene was amplified from pCEP4 (Invitrogen) by PCR with two primers containing ClaI and XhoI sites at the 5Ј and 3Ј ends, respectively.…”

“…Transfection of COS-7 cells was performed as previously described (14). For HIV-1 protease inhibitor experiments, transfected COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 20 M saquinavir until cell lysis and protein analysis.…”

“…For HIV-1 protease inhibitor experiments, transfected COS-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 20 M saquinavir until cell lysis and protein analysis. HeLa cells were also transfected by the DEAE-dextran method, as previously described (14) except that 200 g of DEAE-dextran per ml was used for transfection.…”

“…Protein analysis and sera. Immunoblotting of cell lysates or extracellular viral particles was performed as previously described (14). An HIV-1-positive human serum was obtained from an HIV-1-infected patient from Baltimore, Md.…”