Construction and characterization of Moloney murine leukemia virus mutants unable to synthesize glycosylated gag polyprotein.

Abstract

Retroviruses contain three genes for replication: gag, pol, and env, which encode polyprotein precursors for the internal capsid proteins, reverse transcriptase, and envelope glycoprotein, respectively (1). Murine leukemia virus (MuLV) differs from most other retroviruses in that it encodes two different pathways for gag gene expression (2). These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3-5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as 4-6 kilodaltons (kDa) of amino-terminal protein (6, 7). The additional amino-terminal peptides result in glycosylation of gPr8O'ag during translation (8, 9). gPr8 gag is processed by the further addition of carbohydrate and exported to the cell surface where it appears as a glycoprotein of 95 kDa (8-11). It may be also be released into the extracellular medium as cleavage products of 55 and 40 kDa (8,12). Glycosylated gag products are not incorporated into MuLV virions, but they associate with the extracellular matrix (13).While the function of Pr65gag is known, the role of gPr809ae in MuLV infection is not known. Glycosylated gag might provide some function required for viral replication, or it might play an accessory role. We previously isolated mutants of Moloney MuLV (M-MuLV)-infected mouse fibroblasts that did not express gag proteins at the cell surface, and they were deficient for virus production. However, these mutants were cellular, not viral, in nature and they produced normal amounts of gPr8g'ag intracellularly (14). To obtain a more definitive answer, we constructed two mutants of M-MuLV at the recombinant DNA level and recovered virus by transfection. The constructions. and characterizations of the mutant viruses are described here.MATERIALS AND METHODS Cells and Viruses. All cells were grown in Dulbecco-modified Eagle's medium/10% calf serum; Mouse NIH-3T3 cells were described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).The UV-XC assay for MuLV was carried out as described (17). Assay of viral reverse transcriptase by the addition of exogenous poly(rA):oligo(dT) template-primer (16) and banding of virus in sucrose density gradients (18) was carried out as described.Recombinant DNA Cloning. A phage recombinant DNA clones (A-MLV clones 48, 61, and 63) carrying integrated copies of M-MuLV provirus were described previously (19). pMSV-1 is a plasmid clone of unintegrated Moloney murine sarcoma virus (M-MSV) DNA permuted about the unique HindIII site (20). pSLT is a subclone of pMSV-1 deleted from the Sal I site in M-MSV DNA to the Sal I site in the pBR322.vector (see Fig. 2) and was kindly provided by Inder Verma. P90(Abl), a plasmid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain) DNA, was kindly provided by Owen Witte. Plasmid vector pBR328 has been described by Soberon et al. (21).Restriction ...

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex

“…Glyco-Gag has long been known to be important for MLVinduced pathogenesis, and its abrogation leads to decreased virus replication and pathogenesis in vivo (4,(9)(10)(11)(12). However, previous studies carried out primarily in murine fibroblast lines such as NIH 3T3 that do not express APOBEC3 suggested that glyco-Gag was dispensable for in vitro infection.…”

“…To determine whether this was also the case for M-MLV, we infected C57BL/6, BALB/c, and APOBEC3 KO pups with WT and glyco-Gag mutant M-MLV; the APOBEC3 null allele is present on the BL/6 background (26). The mutant virus has a stop codon in the gPr80Gag reading frame (UAU to UAG) at nt 608, 12 nt 5′ of the normal Gag AUG (9). BL/6 mice infected with the glyco-Gag mutant virus showed significantly lower levels of infectious virus in spleens and thymi (Fig.…”

“…Virus was isolated from WT and glyco-Gag mutant M-MLV harvested from NIH 3T3 fibroblasts stably infected with WT and glyco-Gagnegative Ab-X-M-MuLV, respectively, as previously described (9) or from in vivo-infected splenocytes and thymocytes. Infected mice were killed at 16 dpi.…”

“…The conservation of glyco-Gag in many gammaretroviruses suggests that it plays a significant role in their replication. Indeed, although mutations of glyco-Gag do not affect virus replication in vitro (9)(10)(11), they lead to lower in vivo infectivity (9,12,13). Moreover, in mice inoculated with glyco-Gag-deficient M-MLV, there is reversion to WT virus (10, 12, 13).…”

Murine leukemia virus glycosylated Gag (gPr80 gag ) facilitates interferon-sensitive virus release through lipid rafts

“…gPr80 gag 's is conserved among gammaretroviruses, but its mechanism of action has been elusive. MuLVs mutant in gPr80 gag are replication-competent with minor reductions in infectivity in vitro (6). However, when infected into mice, there is strong in vivo selection for recovery of gPr80 gag expression (7)(8)(9).…”