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Journal of Virology, December 2009, p. 12336-12344, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00676-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutations in the Thumb Allow Human Immunodeficiency Virus Type 1 Reverse Transcriptase To Be Cleaved by Protease in Virions {triangledown}

Linda L. Dunn,1 Mary Jane McWilliams,1 Kalyan Das,2 Eddy Arnold,2 and Stephen H. Hughes1*

HIV-Drug Resistance Program, NCI-Frederick, Frederick, Maryland 21701,1 Rutgers University, Department of Chemistry and Chemical Biology, Piscataway, New Jersey 088542

Received 1 April 2009/ Accepted 4 September 2009

Although human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been extensively studied, there are still significant questions about the effects of mutations on the maturation and stability of RT. We show here that a significant fraction (>80%) of the single point mutations we generated in the thumb subdomain of HIV-1 (RT) affect the stability of RT in virions. Fragments of the unstable mutant RTs can be detected in Western blots of virion proteins; however, the degree of degradation varies. The titers of the mutants whose virions contain degraded RTs are reduced. Some, but not all, of the unstable RT thumb subdomain mutants we analyzed have a temperature-sensitive phenotype. A preliminary survey of mutations in other subdomains of RT shows that some of these mutations also destabilize RT. The stability of the RT mutants is enhanced by the addition of a protease inhibitor, suggesting that the viral protease plays an important role in the degradation of the mutant RTs. These results confirm and extend earlier reports of mutations that affect the stability of RT in virions. The data suggest that the stability of a mutant RT in virions could be a major factor in determining the virus titer and, by extension, viral fitness, which could affect whether a mutation in RT is acceptable to the virus.

* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI, P.O. Box B, Bldg. 539, Rm. 130A, Frederick, MD 21702-1201. Phone: (301) 846-1619. Fax: (301) 846-6966. E-mail: hughes{at}ncifcrf.gov

{triangledown} Published ahead of print on 16 September 2009.

Journal of Virology, December 2009, p. 12336-12344, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.00676-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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