Transformation of Mouse 3T3 Cells by Murine Sarcoma Virus: Release of Virus-Like Particles in the Absence of Replicating Murine Leukemia Helper Virus

Cultured cells of different chemicallyinduced C57BL/6N murine sarcomas produced variable amounts of infectious murine leukemia virus (MuLV) and contained proportional amounts of MuLV structural components as determined by radioimmunoassay. Monospecific antisera directed against the major MuLV glycoprotein (gp7l), the major internal antigen (p30), and the ribonucleoprotein (plO) were capable of mediating tumor cell lysis in the presence of complement, suggesting that these viral structural components were localized at least in part to the cell surface. Membrane immunofluorescence studies with MuLV p30 antiserum confirmed surface localization. Addition of MuLV p30 polypeptide to normal cells and tumor cells enhanced the cytotoxicity of MuLV p30 antiserum. Studies are presented which suggest that the presence of MuLV structural components on cell surfaces can be independent of virus production and cellular transformation.

Section: Methodsmentioning

confidence: 99%

Expression of Murine Leukemia Virus Structural Antigens on the Surface of Chemically Induced Murine Sarcomas

Grant

Bigner

Fischinger

et al. 1974

“…The gag gene product is apparently able to assemble into virus particles in the absence of other virus components (refs. [1][2][3][4]; A.R. and A.M.S., unpublished results).…”

mentioning

confidence: 99%

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

Rein¹,

McClure²,

Rice³

et al. 1986

270

211

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

“…Retroviral gag protein alone can oligomerize and assemble into virus-like particles, both in vitro and in vivo (1)(2)(3)(4)(5)(6)(7)(8)(9), even in the absence of genomic RNA, RT, viral protease, or envelope (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Similarly, gag particle assembly can occur not only intracellularly but even in an in vitro translation system (2).…”

mentioning

confidence: 99%

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

Sharma

Murai²,

Miyanohara³

et al. 1997

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted͞budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 10 5 colony-forming units per ml.The role in assembly of two retrovirus-encoded proteins, gag and env, have been extensively studied and are reasonably well understood. The retrovirus gag protein is central to the mechanisms of virus assembly because of its ability to interact with both the viral RNA and with the virus envelope proteins. In the case of the C-type retroviruses, as represented by Moloney murine leukemia virus (MoMLV), virion assembly and maturation include intracellular oligomerization of gag, its association with viral RNA via a recognition of the packaging signal ⌿ on the RNA, and the subsequent specific interaction of the resulting complex with plasma membrane-embedded env protein. In addition to viral RNA, env, and gag, a number of other viral components, including the virus-encoded pol gene products protease, reverse transcriptase (RT), and integrase, participate in the full assembly of mature fully packaged and infectious virus particles. The assembly of virus-like particles, however, does not require the presence of all these components. Retroviral gag protein alone can oligomerize and assemble into virus-like particles, both in vitro and in vivo (1-9), even in the absence of genomic RNA, RT, viral protease, or envelope (10-20). Similarly, gag particle assembly can occur not only intracellularly but even in an in vitro translation system (2). In addition, expression of HIV gag and pol genes results in the production of virus-like particles that contain gag protein and RT activity and that associate with virus RNA molecules, even in the absence of virus env (4). The role, if any, that these particles play in the final assembly of mature virus particles is unknown.In addition to virus assembly, the env protein performs at least two other important viral functions: a receptor recognition function that allows the specific binding of the virus to a cell surface receptor required for uptake into an endosome and a fusion function that permits the release of the uncoated virus core and the viral RNA from the endosome into the cytoplasm. Through its interaction with specific cellular receptors, the env glycoprotein of each retrovirus defines the tropism of the virus. Rec...