Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors

A sensitive and quantitative cell-free infection assay, utilizing recombina nt human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the viru s have prevented a clear understanding of these events. Virus stocks were g enerated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfe r vector containing either firefly luciferase or enhanced yellow fluorescen t protein genes, and (iii) an envelope expression plasmid. Single-round inf ections were initiated by exposing target cells to filtered supernatants an d quantified by assaying for luciferase activity in cell extracts or by enu merating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as s hown by the effects of the reverse transcriptase inhibitor 3 ' -azido-3 ' - deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determin ed to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 p articles, pseudotyped with either vesicular stomatitis virus G protein or H TLV-1 envelope, was typical of retroviruses, exhibiting a half-life of appr oximately 3.5 h at 37 degreesC. The specific infectivity of recombinant HTL V-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus , the low infectivity of HTLV-1 is determined in large part by properties o f the core particle and by the efficiency of postentry processes.