Human immunodeficiency virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells.
BACKGROUND
HIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells.


RESULTS
Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p66(8.44) and p51(8.31)). The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs), and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p66(8.53) and p51(8.60)), suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p66(8.44) and p51(8.31) differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay.


CONCLUSIONS
The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.
Davis
Adam J
AJ
Infectious Diseases Laboratories, SA Pathology, Adelaide 5000, Australia. adam.davis@adelaide.edu.au
Carr
Jillian M
JM
Bagley
Christopher J
CJ
Powell
Jason
J
Warrilow
David
D
Harrich
David
D
Burrell
Christopher J
CJ
Li
Peng
P
D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D007526:Isoelectric Point; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D014771:Virion / Q000737:chemistry*
D002457:Cell Extracts; D020033:Protein Isoforms; D020543:Proteome; D012194:RNA-Directed DNA Polymerase