Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

József Tözsér,Michael S. Mitchell,Ashleigh Auth,Gerald Princler,David Derse,Patricia A. Lloyd

doi:10.1074/jbc.m507660200

Abstract

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Highlights

Human T-cell lymphotropic virus type-1 (HTLV-1)2 is an oncogenic retrovirus belonging to the deltaretrovirus genus, which includes HTLV-2, -3, and -4, simian T-cell lymphotropic viruses, and bovine leukemia virus
Pol Synthesis and Predicted Cleavage Products—Expression of HTLV-1 Pol requires that two ribosomal frameshifts occur during translation of viral mRNA
If cleavage were to occur between the polymerase and RNase H domain in HTLV-1 reverse transcriptase (RT), similar to the cleavage that takes place in human immunodeficiency virus (HIV)-1 RT, the resulting peptides would be ϳ50 and 14 kDa, respectively

Summary

Introduction

Human T-cell lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus belonging to the deltaretrovirus genus, which includes HTLV-2, -3, and -4, simian T-cell lymphotropic viruses, and bovine leukemia virus. The expression of Gag-Pol polyprotein in these retroviruses involves two ribosomal frameshifts: one where gag and pro overlap and the second within the pro/pol overlap [3,4,5,6,7,8,9,10,11]. Based on in vitro translation of viral RNA, the molar ratio of the Gag, Gag-Pro, and Gag-Pro-Pol polyproteins was estimated to be about 100:10:1 [12] This means that the relative amounts of Pol to Gag produced by HTLV-1 would be 5–10-fold less than the amount present in singleframeshift retroviruses, such as HIV and RSV [1, 2] and 3– 4-fold less than the amount found in the other two-frameshift retroviruses: mouse mammary tumor virus and Mason-Pfizer monkey virus [13,14,15]. Immunoprecipitation of an epitope-tagged version of p62 RT from HTLV-1 VLPs co-precipitated the untagged p49 polymerase subunit, suggesting that the virion-associated HTLV-1 RT can exist as a p62/p49 heterodimer, analogous to the HIV-1 p66/p51 RT complex

Methods

Results

Conclusion