Improvement of HIV-1 and Human T Cell Lymphotropic Virus Type 1 Replication-Dependent Vectors via Optimization of Reporter Gene Reconstitution and Modification with Intronic Short Hairpin RNA

Abstract

Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the introncontaining RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cellfree infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection. IMPORTANCEThe human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase-or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay.

The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1)

“…Using this system, our results indicate that Fascin is a major contributor of HTLV-1 cell-to-cell transmission independent of the cell type and the envelope type tested. Despite recent work showing that the reporter system we used even underestimates cell-to-cell transmission events [ 55 ], we still see a significant reduction of reporter gene activity reflecting cell-to-cell transmission upon repression of endogenous or Tax-induced Fascin expression. However, new reporter vectors with improved splicing and packaging of the spliced reporter RNA might allow for better quantitating the role of Fascin on cell-to-cell transmission in lymphocytes and in primary cells in future work [ 55 ].…”

Molecular Mechanisms of HTLV-1 Cell-to-Cell Transmission

“…One obstacle when working with these reporter vectors was the lack of sufficient reporter signals in PBMC [ 57 ]. Recently, Mazurov and colleagues improved the reporter vectors by modifying the splice sites, and by enhancing packaging efficiency of spliced reporter vectors [ 108 ]. It will be interesting to see, which Tax-induced signaling pathways and host factors are required for viral transmission to PBMC.…”

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method

“…Three days postinfection, the transduced cells were selected by growing in the presence of puromycin (Sigma, St. Louis, MO, USA) at a concentration increasing from 0.4 to 1.0 µg/mL. The cell coculture infections were performed as described earlier [ 19 , 20 ]. Briefly, to set up HIV-1 infection, 10 6 CEM cells were electroporated with 3 µg of pUCHR-inLuc-mR vector DNA, 2 µg of pCMV∆8.2R plasmid DNA, and 0.8 µg of the pIIINL-4env plasmid, which expresses Env from HIV-1 strain NL4-3.…”