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Related Experiment Videos

Construction and characterization of a temperature-sensitive human immunodeficiency virus type 1 reverse

M Huang1, R Zensen, M Cho

  • 1Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.

Journal of Virology
|March 14, 1998
PubMed
Summary

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A temperature-sensitive human immunodeficiency virus type 1 reverse transcriptase mutant showed impaired virion production at higher temperatures. This defect is linked to aberrant processing of the reverse transcriptase during maturation within new virus particles.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is crucial for viral replication.
  • Understanding the molecular mechanisms of RT function and its potential temperature sensitivity is important for antiviral strategies.

Purpose of the Study:

  • To generate and characterize a temperature-sensitive (ts) mutant of HIV-1 RT.
  • To investigate the impact of specific mutations in the RT finger domain on viral replication and particle production.

Main Methods:

  • Charged-cluster-to-alanine mutagenesis was used to create the ts RT mutant (K64A, K66A, D67A).
  • Virus replication was assessed at permissive (34.5°C) and non-permissive (39.5°C) temperatures.
  • Virus particle production was quantified using p24 antigen capture and virion-associated RT activity.

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  • Virus infectivity was measured by the MAGI cell assay.
  • Radioimmunoprecipitation and Western blot analyses were performed to examine RT subunit presence and Gag-Pol processing.

    • Main Results:

    • The mutant virus replicated normally at 34.5°C but not at 39.5°C.
    • Virions produced at the permissive temperature were functional at the non-permissive temperature, indicating the RT enzyme itself was not ts.
    • Virus particle production at the non-permissive temperature was comparable to wild-type, but particle-associated RT activity and infectivity were significantly reduced.
    • Absence of p66 and p51 RT subunits in virions produced at 39.5°C was observed.
    • Normal levels of HIV-1 integrase and Gag-Pol precursor were detected, ruling out defects in their synthesis or incorporation.
    • Mutant Pr160(gag-pol) was detected in virions when HIV-1 protease was inactivated.

    Conclusions:

    • The ts defect is irreversible and occurs during virus particle production, specifically affecting RT processing or maturation.
    • The results suggest a critical role for proper RT processing/maturation within nascent particles for viral infectivity.
    • The findings point towards a potential defect in the degradation or aberrant processing of the mutated RT during its maturation within developing virions.