Human T-Cell Leukemia Virus Type 1 Reverse Transcriptase (RT) Originates from the pro and pol Open Reading Frames and Requires the Presence of RT-RNase H (RH) and RT-RH-Integrase Proteins for Its Activity

Abstract

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH–RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the α3/β type.

Infectious transmission and replication of human T-cell leukemia virus type 1

“…We also showed that the addition of 8 amino acids to the aminoterminus of RT abolished polymerase activity. The carboxy-terminus of RT is identical to that predicted from amino acid sequence alignments, reported previously (21). In addition to RT and IN, we detect smaller Pol cleavage products that contain either polymerase or RNaseH domains.…”

Human Immunodeficiency Virus Type 1 Integrase Protein Promotes Reverse Transcription through Specific Interactions with the Nucleoprotein Reverse Transcription Complex

“…Our findings that show a direct physical interaction between the IN and RT proteins strongly suggest that the IN protein is directly involved in reverse transcription in vivo. In the case of the avian retroviruses, the IN protein comprises an integral component of the RT heterodimer; an RT-IN polypeptide makes up the beta subunit (30,50). Taken together, these results suggest that the IN protein forms an integral part of the reverse transcription initiation complex and specifically promotes interactions between RT, the genomic RNA, and primer tRNA 3…”

“…One explanation for the effect of IN on reverse tran-scription is that IN affects RT via a direct physical interaction. Several observations suggest that HIV RT and IN may form a heterodimeric complex: (i) the two proteins are known to coexist as a complex in some retroviruses (30,50), (ii) the car- a Four micrograms of DNA of each of the viral clones was transfected into 293T cells, either alone (Ϫ) or together with 2 g of the pLR2P-vprIN expression plasmid (ϩ), by calcium phosphate DNA precipitation methods. Forty-eight hours later, the culture supernatants were harvested, clarified by low-speed centrifugation, filtered through 0.45 m-pore-size filters, and saved as stocks.…”

Characterization of human T‐cell leukemia virus type I integrase expressed in Escherichia coli

“…This species could correspond to either a monomer comprising both the RT and the IN homology region, similar to the β subunit of avian sarcoma‐leucosis virus RT [31], or a dimer of RT‐derived subunits, analogous to HIV RT [32]. Recently it has been suggested that HTLV RT may be an oligomer consisting of RT and RT‐IN subunits [32a]. From amino acid sequence comparisons, it is likely that the mature HTLV‐I IN consists of the 300 C‐terminal amino acids of Pol (from Pro597 to the C‐terminus).…”