Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

Abstract

HIV-1 virions contain two reverse transcriptase polypeptides that have apparent molecular weights of 66 and 51 kDa. The 51 -kDa form lacks the carboxy-terminal sequences found in the 66-kDa form, and is believed to be a proteolytic digestion product. We have treated purified 66-kDa reverse transcriptase with viral and nonviral proteases. The digestion products were characterized by their ability to react with monoclonal antibodies known to recognize particular segments of the HIV-1 reverse transcriptase.The approximate location of the segments recognized by the monoclonal antibodies was determined by testing the ability of the antibodies to recognize a series of amino-and carboxy-terminaldeleted forms of HIV-1 reverse transcriptase.The segments recognized are not uniformly distributed along the primary amino acid sequence of HIV-1 reverse transcriptase.We suggest that these segments are probably on the surface of the properly folded form of reverse transcriptase.Of the tested proteases, only the viral protease was able to cleave the 66-kDa form to the 51-kDa form without producing additional cleavage products, suggesting that the viral protease cleaves the 66-kDa protein to the 51 -kDa form in virions.

DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity

“…Because protease-defective viruses lack the virus-encoded enzyme that cleaves and processes pl60 g"a-~°~, this suggests that mature RT (p66/p51) is involved in the synthesis of the DNA found in virus particles. The very low levels of DNA in protease-defective particles could be synthesized by p160 °"q-~°~ or by traces of p66/p51 or p66/p66, processed from p 160 by cellular proteases (Ferris et al, 1990;Lowe et al, 1988). Viral DNA could be made by RT in the cytoplasm during viral formation, in the immature virion prior to budding, or in the virion after budding.…”

“…The human immunodeficiency virus (HIV) contains two genomic RNA strands in its core, in association with the viral reverse transcriptase (RT) enzyme (Ferris et al, 1990;Lowe et al, 1988;Restle et al, 1990) and primer tRNA lys~ (Jiang et al, 1992;Kleiman et al, 1991). Reverse transcription of the genomic RNA template to form proviral dsDNA is thought to occur in the host cell cytoplasm after core entry (Weiss et al, 1985).…”

“…Reverse transcription of the genomic RNA template to form proviral dsDNA is thought to occur in the host cell cytoplasm after core entry (Weiss et al, 1985). RT exists in the virion as a heterodimer consisting of two subunits of 66K and 51K (Ferris et al, 1990;Larder et al, 1987;Lowe et al, 1988). This form of RT, p66/p51, binds to tRNA lys3 (Barat et al, 1989(Barat et al, , 1991Richter-Cook et aL, 1992;Sarih-Cottin et al, 1992) to form a transcriptional complex on the primer binding site (PBS; Barat et al, 1989Barat et al, , 1991.…”

Immunological Characterization Of The Human Immunodeficiency Virus Type 1 Reverse Transcriptase Protein By The Use Of Monoclonal Antibodies

“…These proteins are an 18K protein with protease activity, a 66K reverse transcriptase (RT) protein and a 34K protein with endonuclease activity (Di Marzo Veronese et al, 1986;Lightfoote et al, 1986;Farmerie et al, 1987;Larder et al, 1987a, b;Mous et al, 1988;Hizi et al, 1990). The 66K RT protein encoded by the middle portion of the pol gene consists of 560 amino acids and it has both RT and RNase H enzymic activities (Hizi et al, 1987(Hizi et al, , 1990Larder et al, 1987a, b;Le Grice et al, 1988).…”

“…These proteins are an 18K protein with protease activity, a 66K reverse transcriptase (RT) protein and a 34K protein with endonuclease activity (Di Marzo Veronese et al, 1986;Lightfoote et al, 1986;Farmerie et al, 1987;Larder et al, 1987a, b;Mous et al, 1988;Hizi et al, 1990). The 66K RT protein encoded by the middle portion of the pol gene consists of 560 amino acids and it has both RT and RNase H enzymic activities (Hizi et al, 1987(Hizi et al, , 1990Larder et al, 1987a, b;Le Grice et al, 1988). The viral protease cleaves the 66K RT protein into an N-terminal 51K and a C-terminal 15K protein which are found in virions in equimolar amounts to the uncleaved 66K protein (Di Marzo Veronese et al, 1986;Farmerie et al, 1987;Lightfoote et al, 1986;Hansen et al, 1988;Mous et al, 1988;Ferris et al, 1990).…”