We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction. Cell-free virus particles released from infected cells can be transmitted to target cells by diffusion or may be conveyed directly to target cells via specific intercellular contacts; the latter is referred to as cell-to-cell infection. Microscopic imaging has shown how viral proteins and virus particles move within and between cells, accumulating at sites of cell-cell contact. While we suspect that these images represent virus infection, it has been difficult to accurately quantify virus replication and provirus formation in most cell-to-cell infection experiments. Retroviral vectors that encode reporter proteins have been invaluable tools for analyzing retrovirus replication and restriction, but they have had limited utility in cell-to-cell infection studies due to high background noise resulting from reporter expression in the producer cells. We report the construction and characterization of retroviral vectors that express reporter protein exclusively in target cells and only after completing a full replication cycle. We have validated this approach and have begun to analyze cell and virus determinants for cell-to-cell infection with vectors for two human retroviruses that infect T cells. We show that the mechanism of transmission and ensuing virus replication depend on the particular virus, the effector and target cell types, and on the specific type of cell-cell interaction.

Tetraspanins interact with a wide variety of transmembrane and intracellular proteins called molecular partners, and modulate their function. In this article, we describe a new partner of tetraspanin web, membrane metalloprotease CD10, which is selectively associated with CD9. By constructing chimeras between tetraspanins CD9 and CD82 (the latter does not interact with CD10) or by using site‐directed mutagenesis, we determined that a portion of the large extracellular loop from the CCG motif to transmembrane domain 4, as well as the C‐terminal tail of CD9, are involved in the interaction with CD10. The stable expression of wild‐type CD9 in K562 CD10‐positive cells enhanced the level of CD10 released with exosomes five‐fold. In contrast, the expression of chimeric CD9, which contained the cytoplasmic C‐terminal domain from CD82, had little effect on CD10 release. Short hairpin RNA knockdown of CD9 expression in Nalm‐6 pre‐B cells resulted in a two‐fold reduction in the amount of endogenous CD10 released with microvesicles. The peptidase activity of CD10 measured either on cells or on exosomes correlated with the level of CD10 expression, and was not significantly modulated by CD9 expression as such. Our data suggest that the interaction of CD10 with tetraspanin CD9 can play an important role in the redistribution of peptidase activity from the cell surface to outer microenvironments. In bone marrow, where CD10 presumably contributes to the maturation of pre‐B cells and migration of B cells to the blood circulation, release of CD10 peptidase activity with exosomes may effectively regulate extracellular matrix microenvironments.Structured digital CD9 physically interacts with CD179b, Tetraspanin-9, CD63, CD81, HLA class II, Uncharacterized protein C14orf166B, Tetraspanin-14, HLA class I, Galactokinase, Protein disulfide-isomerase A6, CD98, CD19, CD316, CD146, CD92, Annexin A6, CD156c, CD71, CD10, CD29, CD315, DAAM1 and CD49f by anti bait coimmunoprecipitation (View interaction) CD10 physically interacts with CD9 by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3, 4) CD10 physically interacts with CD179b, Protein FAM207A, CD9, CD81, Uncharacterized protein C22orf13, HLA class II, Uncharacterized protein C3orf26, HLA class I, Protein C14orf21, Annexin A6 and DAAM1 by anti bait coimmunoprecipitation (View interaction)

So far, only two retroviruses, human immunodeficiency virus (HIV) (type 1 and 2) and human T-cell lymphotropic virus type 1 (HTLV-1), have been recognized as pathogenic for humans. Both viruses mainly infect CD4+ T lymphocytes. HIV replication induces the apoptosis of CD4 lymphocytes, leading to the development of acquired immunodeficiency syndrome (AIDS). After a long clinical latency period, HTLV-1 can transform lymphocytes, with subsequent uncontrolled proliferation and the manifestation of a disease called adult T-cell leukemia (ATLL). Certain infected patients develop neurological autoimmune disorder called HTLV-1-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP). Both viruses are transmitted between individuals via blood transfusion, tissue/organ transplantation, breastfeeding, and sexual intercourse. Within the host, these viruses can spread utilizing either cell-free or cell-to-cell modes of transmission. In this review, we discuss the mechanisms and importance of each mode of transmission for the biology of HIV-1 and HTLV-1.

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies (MAbs) against Jurkat T cells and selected several IgM MAbs that strongly inhibited HTLV-1 but not human immune deficiency virus type 1 (HIV-1) cell-to-cell infection. These MAbs recognized the so-called Tn antigen (GalNAcα1-O-Ser/Thr) that arises on Jurkat cells from a mutation in the T-synthase-specific chaperone Cosmc and the consequent loss ofO-glycan elongation. Anti-Tn MAbs precipitated two majorO-glycan carrier proteins, CD43 and CD45, and caused a strong aggregation of Jurkat cells. The restoration of O-glycosylation in Jurkat cells by stably transducing the wild-typeCosmcgene resulted in a 3- to 4-fold increase in the level of surface expression of CD43 and enhanced HTLV-1 transmission 10-fold in comparison to that of parental cells. The short hairpin RNA (shRNA) knockdown of CD43 or CD45 expression in Jurkat-Cosmc, HBP-ALL, and CEM T cells decreased HTLV-1 infection severalfold. The knockdown of CD45 in Jurkat cells severely reduced both HTLV-1 and HIV-1 infections, but Cosmc coexpression partially rescued infection. HTLV-1 proteins, which assembled in small patches on Jurkat cells, formed large clusters on the surface of Jurkat-Cosmc cells. These data indicate that large aggregates of HTLV-1 assemblies are more infectious than multiple clustered virions. We suggest that heavily O-glycosylated CD43 and CD45 molecules render cells less adhesive, prevent inappropriate cell-cell contacts, and favor the assembly of HTLV-1 particles into large, highly infectious structures on the surface of T cells.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

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 intron-containing 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 cell-free 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. IMPORTANCE The 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.