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 degrees 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.

FIG. 1

HTLV-1 packaging plasmids and transfer vectors. pCMV-HT1 was constructed from an infectious molecular clone, pCS-HTLV, by replacing the 5′ LTR, tRNA primer binding site, and RNA encapsidation elements with a CMV promoter joined to the major splice donor site from the HTLV-1 R region. A XhoI fragment was deleted from the env gene of pCMV-HT1 to produce pCMVHT-Δenv. HTLV-1 transfer vectors were constructed by replacing sequences between the gag and pX genes in pCS-HTLV with promoter/reporter gene cassettes. pHTC-luc contains a CMV promoter joined to the firefly luciferase gene. pHTC-luc-tsa was made by inserting a fragment that contains the splice acceptor site (SA) from the third exon of the tax/rex gene immediately upstream of the CMV promoter into pHTC-luc. Analogous transfer vectors were constructed in which the luciferase gene was replaced with the eYFP gene to give pHTC-eYFP and pHTC-eYFP-tsa.

FIG. 2

AZT inhibits HTLV-1-mediated gene transduction. Recombinant virus was generated from 293 cells transfected with pHTC-luc, pCMVHTΔenv, and pCMV-VSV-G. Human 293T cells were treated with no drug or with 3, 10, 30, 100, or 500 nM AZT for 3 h prior to infection and then maintained in AZT until lysis at 72 h postinfection. Luciferase activities are expressed relative to the no-drug control. The data are the averages of two experiments.

FIG. 3

Luciferase transduction is proportional to the number of infected cells. Human 293T cells were infected with serial twofold dilutions of virus generated from cells transfected with HTLV-1 vectors pCMVHT-Δenv, pCMV-VSV-G, and either pHTC-luc or pHTC-eYFP. In a parallel experiment, cells were infected with serial 10-fold dilutions of filtered supernatants from cells transfected with HIV-1 vectors pCMV-ΔR8.2, pHR′-CMVeGFP, and pCMV-VSV-G. At 72 h after infection, cells infected with pHTC-luc were assayed for luciferase activity, and cells infected with pHTC-eYFP or with pHR′-CMVeGFP were enumerated by flow cytometry. Luciferase activity (relative light units [RLU]) and the number of fluorescent cells are expressed per 10⁶ target cells and are plotted versus the log₁₀ virus dilution factor. The experiment was performed three times, and data from a typical experiment are shown.

FIG. 4

Synthesis and encapsidation of transfer vector mRNAs. (A) mRNAs expressed from pHTC-luc or pHTC-eYFP and pHTC-luc-tsa or pHTC-eYFP-tsa transfer vectors are depicted. The mRNA initiating in the CMV promoter is constitutively expressed and is predicted to be approximately 2.5 kb. The mRNA initiating in the 5′ LTR (LTR/US) is expected to be 4.8 kb and is expressed only in response to trans-regulatory proteins supplied by the packaging plasmid. Transfer vectors containing a splice acceptor site (SA) are predicted to generate an additional spliced mRNA (LTR/S) of 3.5 kb. The transcription pattern for the HIV-1 transfer vector, pHR′-CMVeGFP, is analogous to pHTC-eYFP-tsa, but the mRNAs are smaller. (B) Northern blot analysis of poly(A)⁺ mRNAs extracted from cells transfected with pHTC-eYFP-tsa (lane 1), pHTC-eYFP-tsa plus pCMVHT-Δenv (lane 2), or pHR′-CMVeGFP plus pCMV-ΔR8.2 (lane 4). Virion RNAs were extracted from concentrated virus particles produced by cells transfected with HTLV-1 vectors pHTC-eYFP-tsa plus pCMVHT-Δenv (lane 3) or with HIV-1 vectors pHR′-CMVeGFP plus pCMV-ΔR8.2 (lane 5). The amounts of virion RNA loaded on the gel are equivalent to 7.5 ml of supernatant for recombinant HTLV-1 (lane 3) and 0.75 ml of supernatant for recombinant HIV-1 (lane 5). RNAs were resolved on a 1.2% agarose–formaldehyde gel, transferred to nylon membranes, and hybridized to a ³²P-labeled eYFP probe. Positions of RNA size markers (Ambion Millennium Markers) are indicated. The experiment was performed five times, and representative results are shown.

FIG. 5

HTLV-1 particle stability is typical of oncoretroviruses. The stability of HTLV-1 virions pseudotyped with either VSV-G (solid circles) or HTLV-1 envelope (open circles) was determined by incubating filtered supernatants at 37°C for 0, 2, 4, 6, 8, and 10 h prior to infection of 293T cells. Luciferase activities were determined in cell extracts prepared 72 h after infection and are expressed relative to the no-preincubation control. The data are the averages of two experiments.