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Journal of Virology, November 2005, p. 13694-13704, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13694-13704.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

Kevin W. Chang,1 John G. Julias,1,2 W. Gregory Alvord,3 Jangsuk Oh,1 and Stephen H. Hughes1*

HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland,1 Basic Research Program, SAIC-Frederick, Inc., Frederick, Maryland,2 Data Management Services, National Cancer Institute at Frederick, Frederick, Maryland 21702-12013

Received 2 May 2005/ Accepted 3 August 2005

Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus "PPT." Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3' end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3' A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5' end of the U3. A reciprocal G-to-A substitution at the 3' end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions.

* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI-Frederick, 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.

Journal of Virology, November 2005, p. 13694-13704, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13694-13704.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oh, J., Chang, K. W., Hughes, S. H. (2008). Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA. J. Virol. 82: 11480-11483 [Abstract] [Full Text]  
  • Chang, K. W., Oh, J., Alvord, W. G., Hughes, S. H. (2008). The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase. J. Virol. 82: 8592-8604 [Abstract] [Full Text]  
  • Oh, J., Chang, K. W., Wierzchoslawski, R., Alvord, W. G., Hughes, S. H. (2008). Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate. J. Virol. 82: 503-512 [Abstract] [Full Text]  
  • Oh, J., Chang, K. W., Alvord, W. G., Hughes, S. H. (2006). Alternate polypurine tracts affect rous sarcoma virus integration in vivo.. J. Virol. 80: 10281-10284 [Abstract] [Full Text]  


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