Suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences.
The Tat protein of human immunodeficiency virus type 1 (HIV-1) has been shown to restrict premature reverse transcription at late stages of virus infection and to thus ensure the integrity of the viral RNA genome for packaging. To gain further insights into the roles of Tat in HIV-1 reverse transcription, we have assessed its effects on the first-strand transfer during the synthesis of minus-strand DNA through use of a reconstituted cell-free system. The results demonstrated that a form of Tat, containing only the first exon (Tat72), was able to enhance the first-strand transfer as efficiently as did the viral nucleocapsid protein. Coincidentally, this form of Tat was unable to inhibit the production of minus-strand strong-stop DNA. Further studies with various mutated forms of Tat showed that its Cys-rich region, rather than the core and Arg-rich domains, was essential for this strand transfer activity. Moreover, this activity of Tat is largely independent of the TAR RNA structure. Although full-length Tat protein (Tat86) was also able to promote strand transfer, this activity was limited by a strong overall inhibition of reverse transcription because of the presence of the second Tat exon. Other nucleic-acid-binding proteins (e.g., single-strand DNA-binding protein) were employed as negative controls and were unable to promote strand transfer in these assays. We propose that Tat possesses nucleic acid chaperone activity and can promote the first-strand transfer during HIV-1 reverse transcription; however, these activities are restricted by the second Tat exon, and the roles of these Tat activities in viral replication remain to be elucidated.
Guo
Xiaofeng
X
McGill AIDS Centre, Lady Davis Institute/Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada.
Kameoka
Masanori
M
Wei
Xin
X
Roques
Bernard
B
Gotte
Matthias
M
Liang
Chen
C
Wainberg
Mark A
MA
D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D017931:DNA Primers; D005091:Exons; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D013698:Templates, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D054303:HIV Reverse Transcriptase