trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus

Abstract

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.

Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing

“…A similar phenotype was observed for the MPMV envelope containing a 35-amino-acid deletion in the extracellular domain of TM (3). Similar results have also been observed in other retroviral systems due to changes in SU, not TM (19,33,36,52). Analysis of these mutant precursors suggests that when the block in the envelope processing pathway occurs in the endoplasmic reticulum, the precursor is not transported to the cell surface.…”

Threshold Number of Provirus Copies Required per Cell for Efficient Virus Production and Interference in Moloney Murine Leukemia Virus-Infected NIH 3T3 Cells

“…Cells plated at 5 ϫ 10 5 cells per 6-cm dish were pulselabeled with 60 Ci of L-[ 35 S]methionine in 2 ml of methionine-free minimal essential medium for 60 or 30 min at 37°C and chased in Dulbecco's modified Eagle's medium containing 7% fetal calf serum. The labeled cells were lysed with 800 l of RIPA buffer, and 400 l of each sample was subjected to immunoprecipitation with the anti-gp70 antibody as previously described (19). The immunoprecipitated proteins were electrophoresed through an SDS-10% polyacrylamide gel and exposed to Amersham Hyperfilm-MP X-ray film.…”

Interaction between the dominant negative mutant and the wild-type envelope proteins of Friend murine leukemia virus

“…We previously reported a dominant negative env mutant, referred to as fcr, of Friend murine leukemia virus (FMLV) (19). The fcr mutant env had a point mutation which resulted in a Cys (C)-to-Arg (R) substitution at the 361st amino acid in the FMLV envelope protein (Env).…”

“…The SU-TM, if folded correctly, is multimerized in the ER and transported to the Golgi complex, where it is cleaved into the surface protein (SU, gp7O) and the transmembrane protein (TM, pl5[E]), which are transported to the cell surface (9,15). When the wild-type Env was coexpressed with fcr, the processing of the wild-type Env was inhibited at the ER and the processed Env (SU) became undetectable (19).…”

“…The fcr gene tagged with Lck was introduced into the Moloney MLV (MMLV) long terminal repeat-driven env expression plasmid containing a neomycin-resistant marker gene ( Fig. 1) (19). This Lck-tagged fcr expression plasmid, pNeFCrL, and the wild-type and fcr mutant FMLV env expression plasmids, pHmFW and pHmFCr, containing a hygromycin B-resistant gene (19) were transfected separately into NIH 3T3 cells by the calcium phosphate method (22) to obtain a G418-resistant cell clone, FCrL1, and hygromycin B-resistant cell clones, FW1 and FCrl, respectively.…”