Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Abstract

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.Murine cells expressing hu-CD4, CCR5, and hu-cyclin T1 support the early steps in the retrovirus replication cycle, including entry, reverse transcription, and integration. Following integration, sufficient quantities of viral mRNA transcripts are synthesized, and the cells accumulate abundant quantities of virion structural proteins. Nevertheless, with the possible exception of Chinese hamster ovary cells, none of the cell lines tested supports efficient virus replication. Although entry, integration, and expression of virus proteins occur efficiently, virion assembly and Gag polyprotein processing are severely impaired, resulting in markedly reduced levels of virus release and the failure of the virus to spread

Matrix-Induced Inhibition of Membrane Binding Contributes to Human Immunodeficiency Virus Type 1 Particle Assembly Defects in Murine Cells

“…Previous studies have shown that replacement of the HIV-1 MA domain with that of MLV MA can improve HIV-1 assembly in murine cells (3,18). However, such chimeric viruses are poorly infectious and no manipulation has yet succeeded in improving the yield of infectious HIV-1 virions from murine cells.…”

“…Gag -membrane interaction, processing, and particle release are enhanced by replacing the HIV-1 matrix (MA) domain with that of murine leukemia virus (MLV) (3,18), and this has been interpreted to suggest that a Pr55…”

Targeting of Murine Leukemia Virus Gag to the Plasma Membrane Is Mediated by PI(4,5)P 2 /PS and a Polybasic Region in the Matrix

“…The MuLV MA protein does not contain an obvious PIP binding site, and poor solubility has thus far precluded high-resolution structural studies of the myristoylated MuLV MA protein. It is possible, however, to replace HIV MA with MuLV MA with limited effects on viral replication, suggesting a conserved function for this domain among retroviruses (10,42). To determine if this evolutionarily distant retrovirus uses a similar PIP-dependent mechanism for membrane targeting of the Gag polyprotein, we prepared and characterized native and mutant forms of MuLV MA mutants.…”

Contribution of Virus-Like Particles to the Immunogenicity of Human Immunodeficiency Virus Type 1 Gag-Derived Vaccines in Mice

“…However, evaluating the immunogenicity of HIV-1 Gag VLPs in mice is complicated by the failure of HIV-1 Gag to efficiently assemble VLPs in murine cells. In this study, the problem was overcome by using an MMLV-HIV-1 chimeric Gag that releases significant amounts of VLPs when expressed in murine cells (13,47). This has permitted an evaluation of the role played by in vivo VLP formation in the immunogenicity of HIV-1 Gag.…”

“…It has been previously demonstrated that murine cells expressing HIV-1 proviral clones in which the p17 matrix domain from HIV-1 Gag was replaced with the p15 matrix and p12 domains from MMLV were able to support plasma membrane localization of Gag in murine cells and release significant amounts of viral particles (13,47). To explore the immunogenicity of such Gag VLPs in vivo, we generated a high-expression form of this construct by fusing p15 and p12 from MMLV Gag in frame with codonoptimized HIV-1 Gag p24, p2, p7, p1, and p6, creating MHGag (Fig.…”