Examining Human T-Lymphotropic Virus Type 1 Infection and Replication by Cell-Free Infection with Recombinant Virus Vectors

Abstract

A sensitive and quantitative cell-free infection assay, utilizing recombinant 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 virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown 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 determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37°C. The specific infectivity of recombinant HTLV-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 of the core particle and by the efficiency of postentry processes.

CRM1, an RNA transporter, is a major species-specific restriction factor of human T cell leukemia virus type 1 (HTLV-1) in rat cells

“…[38]. pHTC-GFP-Luc, a newly developed reporter vector by David Derse's group, encodes a GFP-luciferase fusion protein, otherwise identical to pHTC-Luc-tsa.…”

“…Seventy-two hours later luciferase activity in the infected cells was measured. For AZT inhibition, 100nM of 3'-Azido-3'-deoxythmidine (AZT) (Sigma) was used to treat the infected cells as described [38].…”

“…Since it is difficult to measure the infectivity of HTLV-1 by conventional methods, we applied a reporter virus assay [38], in which the HTLV-1 pseudovirus harboring a luciferase gene is coated with G proteins of vesicular stomatitis virus (VSV), which shows a broad tropism. Luciferase is driven by the strong CMV promoter, and used as a sensitive marker of the gene expression from viral genomes, which have integrated into their host cells.…”

“…Lymphocytes naturally infected with HTLV-1 produce very few cell-free HTLV-1 virions, and one in 10 5 to 10 6 virions is estimated to be infectious [38,39]. The cell-to-cell spread of HTLV-1 is mediated through fusion of the two cell membranes caused by Env proteins [23,47,48].…”

“…We investigated the early steps of the HTLV-1 lifecycle, between entry and transcription, and the late steps, including formation of infectious virus and cell to cell infection. To investigate these steps quantitatively, we used a pseudovirus system [38] and our newly devised cell fusion assay, because the extremely poor infectivity of free HTLV-1 virions [38,39] makes it impractical to evaluate them by conventional virological methods. Here, we show that expression of hCRM1 in rat cells may be sufficient to enhance production of HTLV-1 proteins and infectious viruses at levels similar to those in human cells.…”