Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

Rein, Alan; Benjers, B M; Gerwin, Brenda I.; Bassin, Robert H.; Slocum, Donald R.

doi:10.1128/jvi.29.2.494-500.1979

Cited by 16 publications

(10 citation statements)

References 33 publications

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“…In this report, we have utilized the biological techniques described earlier (34) to rescue the defective ecotropic genome in the clone 23 cells. Virions containing this defective genome are detected by the complementation plaque assay (33) at low levels, but reach titers of 105 CPFU/ ml after superinfection (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…Cells and virus stocks. All cells used in these studies have been described previously (33)(34)(35). Cells were grown in McCoy 5a medium with 10% heatinactivated fetal calf serum, penicillin, and streptomycin.…”

Section: Materlals and Methodsmentioning

confidence: 99%

“…The production of complementation plaqueforming units (CPFU) by clone 23 is strong evidence that the cells contain a defective variant of ecotropic MuLV. However, previous work with other variants of MuLV has demonstrated that the production of CPFU can be increased pol MUTANT OF B-TROPIC MuLV 743 (34). We therefore superinfected clone 23 with either MuLV-amph or Mo 83 (41), a mink cell focusinducing virus-like isolate which can penetrate the ecotropic restriction barrier (15, 41) presumably by virtue of its recombinant glycoprotein (7).…”

Section: Materlals and Methodsmentioning

confidence: 99%

“…In this report, we describe the isolation and characterization of a nonconditional mutant of B-tropic MuLV which is defective in polymerase and synthesizes an altered polymerase molecule from altered precursors. This defective ecotropic genome can be rescued from infected cells by competent, XC-negative helper virus by techniques previously reported (33,34). Analysis of structural proteins and genomic RNA of nonint Present address: Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD 21205. fectious virions indicates that this mutant represents an isolate with a single defect in the polymerase region of the genome.…”

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confidence: 99%

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Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

Gerwin¹,

Rein²,

Levin³

et al. 1979

J Virol

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A nonconditional mutant of B-tropic murine leukemia virus (MuLV), defective in polymerase, has been isolated by cloning chronically infected cells. The cell clone containing the mutant produced virus particles which were noninfectious. However, superinfection of the cells by replication-competent XC-negative viruses resulted in the rescue of virus capable of forming plaques in a modified XC test, termed the "complementation plaque assay" (A. Rein and R. H. Bassin, J. Virol. 28:656-660, 1978). Analysis of the noninfectious virions produced without superinfection demonstrated that they contained only 2 to 5% of the wild-type level of reverse transcriptase activity. Purification of this activity indicated that it was associated with a smaller molecule than that produced by wild-type virus. Cells producing the mutant virions did not contain the gag-pol precursor, Pr1809"'-1°'; however the cells contained proteins of 147K and 114K daltons precipitable with anti-pol serum. All of the normal structural proteins as well as 70S genomic RNA could be detected in the mutant particles. An interference test indicated that a functional ecotropic glycoprotein was synthesized by the mutant. These results indicate that the mutant has a unique defect in the pol gene.

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Section: Discussionmentioning

confidence: 99%

Section: Materlals and Methodsmentioning

confidence: 99%

Section: Materlals and Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

Gerwin¹,

Rein²,

Levin³

et al. 1979

J Virol

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“…During virus replication, the viral polymerase reverse transcriptase (RT) frequently introduces nucleotide changes into the genome that produce variants known as quasispecies. The very high particle-to-infectiousunit ratio typically found in ecotropic retrovirus stocks (24,25) might be due in part to generation of mutant Env. Analysis of these naturally occurring env sequences might lead to identification of important residues in Env.…”

mentioning

confidence: 99%

Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein

Zavorotinskaya

Albritton

1999

J Virol

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Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

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The Genetics of Murine Leukemia Viruses

Goff

1984

Current Topics in Microbiology and Immunology

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Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

Cited by 16 publications

References 33 publications

Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein

The Genetics of Murine Leukemia Viruses

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