Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells.

1978 ◽  
Vol 28 (3) ◽  
pp. 802-809 ◽  
Author(s):  
H Fan ◽  
R Jaenisch ◽  
P MacIsaac
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Virus Production ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Mouse Cells ◽  
Murine Leukemia

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

1998 ◽  
Vol 72 (7) ◽  
pp. 5414-5424 ◽  
Author(s):  
Takashi Odawara ◽  
Masamichi Oshima ◽  
Kent Doi ◽  
Aikichi Iwamoto ◽  
Hiroshi Yoshikura
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Natural Infection ◽  
Mrna Level ◽  
Virus Production ◽  
Threshold Level ◽  
Moloney Murine Leukemia Virus ◽  
Sigmoid Curve ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT The gag-pol readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376–6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for env expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the envmRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Δwt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286–2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.

scholarly journals Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11365-11372 ◽  
Author(s):  
Lilin Lai ◽  
Hongmei Liu ◽  
Xiaoyun Wu ◽  
John C. Kappes
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Specific Interaction ◽  
Proteolytic Processing ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants intrans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

scholarly journals Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection

2006 ◽  
Vol 80 (1) ◽  
pp. 342-352 ◽  
Author(s):  
Andrew Yueh ◽  
Juliana Leung ◽  
Subarna Bhattacharyya ◽  
Lucy A. Perrone ◽  
Kenia de los Santos ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Gene ◽  
Alanine Scanning Mutagenesis ◽  
Viral Dna ◽  
Murine Leukemia

ABSTRACT Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.

scholarly journals Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition

2002 ◽  
Vol 76 (16) ◽  
pp. 8360-8373 ◽  
Author(s):  
David Lim ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Polypurine Tract ◽  
Dna Ends ◽  
Murine Leukemia

ABSTRACT Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged α-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (ΔC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5′ and 3′ viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5′ end.

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