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

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).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. As with many other retroviruses, HTLV-1 uses ribosomal frameshifting to regulate the relative expression of Gag proteins and the viral replication enzymes. In many retroviruses, including human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV), protein synthesis that initiates from the gag start codon gives rise to both Gag and Gag-Pol polyprotein precursors. A ribosomal frameshift site encoded either at the 5Ј-end of pol (HIV) or at the 3Ј-end of gag (RSV) allows the synthesis of the Gag-Pol polyprotein (1, 2). In contrast, the pro gene of some retroviruses, such as HTLV, mouse mammary tumor virus, and Mason-Pfizer monkey virus, is in a separate reading frame from both gag and pol. Thus, 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 (3-11). In these viruses, three polyprotein precursors are synthesized. In HTLV-1, they are Gag (Pr53), Gag-Pro (Pr76), and Gag-Pro-Pol (Pr180). 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 (13-15).Retroviral pol genes encode RT and integrase (IN), which are required for the replication and integration of the viral genome. The Pol precursor is enzymatically cleaved by the viral protease to yield the subunits of the RT complex. The reverse transcriptases of HIV-1, RSV, and murine leukemia virus are t...

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