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Related Experiment Videos

Mutations in the RNase H primer grip domain of murine leukemia virus reverse transcriptase decrease efficiency and

Jean L Mbisa1, Galina N Nikolenko, Vinay K Pathak

  • 1HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.

Journal of Virology
|December 15, 2004
PubMed
Summary

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Mutations in the murine leukemia virus (MLV) reverse transcriptase (RT) RNase H primer grip increase viral mutation rates and deletions. These changes impair viral replication and DNA synthesis fidelity.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • The RNase H primer grip of reverse transcriptase (RT) is crucial for viral DNA synthesis and replication fidelity.
  • Functional equivalents of human immunodeficiency virus type 1 (HIV-1) RT's primer grip exist in murine leukemia virus (MLV) RT.

Purpose of the Study:

  • To investigate the role of the MLV RT RNase H primer grip in viral replication fidelity.
  • To determine the impact of specific mutations in the MLV RNase H primer grip on viral mutation rates and DNA synthesis.

Main Methods:

  • Mutational analysis of the MLV RT RNase H primer grip.
  • Use of beta-galactosidase (lacZ) and green fluorescent protein (GFP) reporter genes to assess viral mutation rates.
  • DNA sequencing of reporter genes to identify mutation types and frequencies.

Related Experiment Videos

  • Quantitative real-time PCR to analyze reverse transcription product efficiency.

    • Main Results:

    • Specific substitutions (S557A, A558V, Q559L) in the MLV RNase H primer grip significantly increased viral mutation rates (2.1- to 3.8-fold).
    • Mutations led to a higher frequency of deletions between the primer-binding site (PBS) and downstream sequences.
    • Mutant RTs showed substantially reduced efficiency in plus-strand DNA transfer.

    Conclusions:

    • The MLV RNase H primer grip is a key determinant of in vivo DNA synthesis fidelity.
    • Mutant RTs with altered primer grips may struggle to process the DNA-RNA junction, leading to error-prone plus-strand transfer and impaired viral replication.