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M-Sec induced by HTLV-1 mediates an efficient viral transmission

['Masateru Hiyoshi', 'Department Of Safety Research On Blood', 'Biological Products', 'National Institute Of Infectious Diseases', 'Tokyo', 'Naofumi Takahashi', 'Joint Research Center For Human Retrovirus Infection', 'Kumamoto University', 'Kumamoto', 'Youssef M. Eltalkhawy']

Date: 2021-12

Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4 + T cells of HTLV-1 carriers, but not of HTLV-1 - individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag.

In the present study, we identified the cellular protein M-Sec as a host factor necessary for de novo infection of human T-cell leukemia virus type 1 (HTLV-1), the causative retrovirus of an aggressive blood cancer known as adult T-cell leukemia/lymphoma. The inhibition or knockdown of M-Sec in infected cells resulted in a reduced viral infection in several culture models and a mouse model. We recently demonstrated a similar role of M-Sec in macrophages infected with another human retrovirus HIV-1, but it has been generally thought that M-Sec is not related to HTLV-1 infection because of the lack of its expression in CD4 + T cells, the major target of HTLV-1. In this study, we revealed that CD4 + T cells of HTLV-1 asymptomatic carriers, but not those of HTLV-1 - individuals, expressed M-Sec, and that the viral protein Tax mediated the induction of M-Sec. Thus, M-Sec is a new and useful tool for further understanding the process of HTLV-1 transmission.

Funding: This study was supported by grants (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (18K19457 to SS and MH, and 18K07155 to MH), a grant from the Japan Agency for Medical Research and Development (AMED) (19fk0410018h0002 to ON and MH), a grant from the SENSHIN Medical Research Foundation (to SS), and a grant from the Astellas Foundation for Research on Metabolic Disorders (to SS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Hiyoshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

HTLV-1 infects at least 5–10 million people worldwide [ 10 ]. The infection is asymptomatic in most cases, but HTLV-1 causes two distinct diseases: an aggressive blood cancer known as adult T-cell leukemia/lymphoma (ATL), and a neurodegenerative condition known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A recent study also demonstrated an increased risk of premature death among HTLV-1-infected individuals, which is independent of ATL and HAM/TSP [ 11 ]. Cell-to-cell infection is recognized as a central route for the transmission of HTLV-1 because the cell-free infection is inefficient [ 12 – 16 ]. Thus, it is important to fully understand the process of cell-to-cell infection.

We recently demonstrated that M-Sec promotes cell-to-cell infection of HIV-1 [ 8 , 9 ]. Small molecule compound that inhibits M-Sec-induced membrane protrusions reduced HIV-1 production in monocyte-derived macrophages [ 8 ]. Moreover, the knockdown of M-Sec retarded HIV-1 production in U87 glioma cells [ 9 ], a widely-used HIV-1 target cell line. As M-Sec inhibition or knockdown reduces membrane protrusions and cell migration in these cells [ 8 , 9 ], M-Sec appears to contribute to the initial phase of HIV-1 transmission by enhancing membrane protrusions and cell migration. However, the role of M-Sec is likely limited to macrophages among the major targets of HIV-1, because M-Sec is expressed in cells of monocytic lineage, but not in CD4 + T cells [ 3 , 8 ]. In fact, the widely used CD4 + T cell line Jurkat was negative for M-Sec expression, which was the case even after productive HIV-1 replication in the cells [ 8 ]. Similarly, M-Sec is thought not to be related to the cell-to-cell transmission of human T-cell leukemia virus type 1 (HTLV-1), another human retrovirus that preferentially infects CD4 + T cells.

M-Sec (also known as TNF-α-induced protein 2, TNFAIP2) is a key regulator of the formation of plasma membrane protrusions including tunneling nanotubes, the F-actin-containing long plasma membrane extensions [ 1 , 2 ], and plays a critical role in initiating the protrusions or extensions [ 3 , 4 ]. Recent studies have also shown that M-Sec enhances migration, invasion, and metastasis of several cancer cells, such as nasopharyngeal carcinoma [ 5 ], breast cancer [ 6 ], and esophageal squamous cell carcinoma [ 7 ]. M-Sec is a cytosolic protein that shares homology with Sec6, a component of the exocyst complex involved in vesicle trafficking [ 3 ]. However, the molecular mechanisms by which M-Sec, which has no known enzymatic activity, regulate membrane protrusions or extensions, and cell motility are largely unknown.

( A ) The CD3 + CD4 + CADM1 + cells in the live cell gate were sorted from PBMCs of an HTLV-1 carrier, cultured with vehicle (DMSO, upper panels) or M-Sec inhibitor (MSec-i, lower panels) for 3 days, and analyzed for Gag (green). The nuclei were also stained with DAPI (blue). Scale bar: 10 μm. ( B ) The sorted CD3 + CD4 + CADM1 + cells of HTLV-1 carriers (#1 and #2) were analyzed as in ( A ). The numbers of puncta or small clusters of Gag per cell are shown (16 cells for each). *p < 0.05.

( A ) The control (Cr)- or M-Sec knockdown (MSec-KD) MT-2 cells, or control cells pre-treated with M-Sec inhibitor for 48 h (Cr/MSec-i) were analyzed for Gag (red). The nuclei were also stained with DAPI (blue). Yellow arrowheads (top left panel) indicate the large Gag clusters. Scale bar: 10 μm. ( B ) Cells were analyzed as in ( A ). Three different fields were randomly selected, and the percentages of the large Gag cluster + cells were quantified (upper). The numbers of small clusters of Gag per cell are also shown (lower, 16 cells for each). *p < 0.05. ( C ) Cells prepared as in ( A ) were analyzed for Gag expression by flow cytometry. The mean fluorescence intensities (MFI) of Gag are shown by setting the value of M-Sec inhibitor-free control cells as 1 (n = 3). n.s., not significant.

( A ) The control (Cr)- or M-Sec knockdown (MSec-KD) MT-2 cells were stained with phalloidin (to visualize F-actin) and analyzed for the surface area (top, 60 cells for each), height (middle, 10 cells for each), or diameter (bottom, 10 cells for each). *p < 0.05. ( B ) The control (Cr)- or M-Sec knockdown (MSec-KD) MT-2 cells were stained with phalloidin (to visualize F-actin) analyzed for membrane protrusions. Control cells pre-treated with M-Sec inhibitor for 48 h were also added (Cr/MSec-i). Three different fields were randomly selected, and the percentages of membrane protrusions + cells were quantified. *p < 0.05. ( C ) The control (Cr)- or M-Sec knockdown (MSec-KD) MT-2 cells were analyzed for migratory activity by using transwell assay. Control cells pre-treated with M-Sec inhibitor (MSec-i) for 48 h were also added. The migration toward SDF-1 (20 or 100 ng/mL) was also assessed. The numbers of cells that migrated through the inserts into lower wells were enumerated by the trypan blue dye exclusion method (n = 3). *p < 0.05.

We next examined how M-Sec contributes to in vitro and in vivo HTLV-1 infection. M-Sec has been reported to enhance the proliferation of several cancer cells [ 6 , 7 ]. However, we did not find any inhibitory effect of M-Sec knockdown on the proliferation of MT-2 cells ( S6A and S8 Figs), which was the case for SLB-1 cells ( S6A Fig ) and macrophage-like RAW264 cells ( S9 Fig ). Instead, M-Sec knockdown in MT-2 cells caused morphological changes, as evidenced by an increase in the surface area and shorter height/longer diameter ( Fig 7A ). M-Sec knockdown or inhibition also reduced plasma membrane protrusions ( Fig 7B ) and the migratory activity of MT-2 cells ( Fig 7C ). If a cell had one or more F-actin + protrusions longer than 2 μm, it was considered protrusion + ( Fig 7B ). The migration of MT-2 cells toward the chemokine SDF-1/CXCL12 was also impaired by M-Sec knockdown or inhibition ( Fig 7C ).

( A ) The un-humanized or humanized mice were inoculated intraperitoneally with irradiated control (Cr)- or M-Sec knockdown (MSec-KD) MT-2 cells. After 4 weeks, the cells in the liver, spleen, bone marrow, or peripheral blood were analyzed for proviral copies by qPCR (n = 4 for each group). The numbers of proviral copies per 100 cells are shown. *p < 0.05. ( B , C ) The humanized mice were inoculated with irradiated MT-2 cells as in ( A ). After 4 weeks, the liver, spleen, or bone marrow was analyzed for human CD45 + or human CD3 + cells by immunohistochemistry (IHC). In ( B ), semi-quantitative scores are shown (n = 4 for each group). *p < 0.05. In ( C ), typical images of human CD3 + cells (brown) in the liver or bone marrow are shown. Scale bar: 50 μm.

To examine how M-Sec contributes to in vivo HTLV-1 infection, we next utilized a mouse model [ 20 ]. When un-humanized immunodeficient mice were inoculated intraperitoneally with control- or M-Sec knockdown (MSec-KD) MT-2 cells, the number of MT-2 cells in the spleen did not differ between the two groups ( S7 Fig ). Under these conditions, the provirus in the tissues, including the spleen, was below the detection limit (Un-humanized; Fig 6A ). However, once humanized immunodeficient mice were used, the provirus became detectable (Humanized; Fig 6A ), indicating de novo infection of reconstituted human T cells. Of note, under these conditions, the number of proviral copies in the liver, spleen, bone marrow, and peripheral blood of the M-Sec knockdown group were lower than that of the control group ( Fig 6A ). In mouse models of HTLV-1 infection, the number of human cells in tissues correlated with that of proviral copies [ 29 , 30 ]. For instance, Percher et al. reported the proliferation of infected human T cells in spleens of MT-2-inoculated humanized mice, the extent of which correlated with the level of plasma viral load [ 30 ]. Consistent with this finding, the number of human CD45 + cells or CD3 + T cells in the liver, spleen, and bone marrow of the M-Sec knockdown group were lower than that of the control group ( Fig 6B and 6C ). These results further support the idea that M-Sec mediates efficient cell-to-cell HTLV-1 infection.

The CD3 + CD4 + CADM1 + cells in the live cell gate were sorted from the PBMCs of HTLV-1 carriers (n = 5). The CD3 + CD4 + cells were also sorted from PBMCs of HTLV-1 − individuals as target cells. They were mixed and co-cultured in the absence (None) or presence of M-Sec inhibitor (MSec-i) or Ral inhibitor (Ral-i) for 2 or 4 days. The number of proviral copies in the co-culture was quantified using qPCR and is shown as percentages relative to that of the co-culture with no inhibitor on day 4 (the third bar from the right). *p < 0.05.

The results of co-cultures using cell lines prompted us to test the effect of the M-Sec inhibitor (MSec-i) and Ral inhibitor (Ral-i) on a primary cell-based co-culture. HTLV-1-infected CD4 + T cells were enriched in the CADM1 + fraction [ 28 ]. When sorted and cultured ( S4A Fig ), approximately half of the CD3 + CD4 + CADM1 + cells of carriers expressed M-Sec at a detectable level, albeit modestly when compared to monocytes ( S4B and S4C Fig ). Thus, we co-cultured the CD3 + CD4 + CADM1 + cells of carriers and the CD3 + CD4 + cells of HTLV-1 - individuals as target cells, and confirmed that both M-Sec inhibitor and Ral inhibitor reduced the number of proviral copies in the co-culture ( Fig 5 ). The timing (day 2 or 4) and extent of the inhibitory effect of those inhibitors were variable among carriers ( S5 Fig ), as observed with the M-Sec induction in CD4 + T cells of carriers ( Fig 1B ). As an obvious increase in the number of sorted CD3 + CD4 + CADM1 + cells during the culture period was not observed in a microscopic analysis, the increase in the number of proviral copies might mainly reflect de novo infection. M-Sec knockdown did not affect the proliferation of MT-2 or SLB-1 cells, and M-Sec inhibitor or Ral inhibitor did not show any cytotoxicity to MT-2, SLB-1, Jurkat or primary CD4 + T cells at the concentration used ( S6 Fig ). Thus, the results of different co-culture systems suggest that M-Sec mediates efficient cell-to-cell HTLV-1 infection.

A group of small GTPases is a possible downstream effector of M-Sec, as both small GTPases and M-Sec regulate actin cytoskeleton remodeling [ 3 , 6 , 25 ]. Among the inhibitors of small GTPases tested [ 26 , 27 ], a Cdc42 inhibitor (ZCL278) reduced viral infection to target cells in MT-2-based co-culture ( Fig 4B , upper), whereas a Ral inhibitor (BQU57) reduced viral infection in both MT-2- and SLB-1-based co-cultures ( Fig 4B ). This result was consistent with the finding that M-Sec-induced membrane protrusions in macrophage-like RAW264 cells were strongly inhibited by a dominant-negative Ral, and modestly inhibited by a dominant-negative Cdc42 [ 3 ].

( A ) Reporter Jurkat cells were cultured alone (Jurkat alone), or co-cultured with the control (Cr) or M-Sec knockdown (MSec-KD) MT-2- or SLB-1 cells for 16 h. In assays in which the effect of M-Sec inhibitor (MSec-i) was tested, control MT-2- or SLB-1 cells were pre-treated with MSec-i for 48 h and used for the co-culture (Cr/MSec-i). The anti-gp46 Env neutralizing antibody was included as a reference (Cr/Env Ab). The luciferase activities are shown by setting the value of Jurkat alone as 1 (n = 3). *p < 0.05. ( B ) The control MT-2- or SLB-1 cells were pre-cultured in the absence (Cr) or presence of Ral inhibitor (Cr/Ral-i), Cdc42 inhibitor (Cr/Cdc42-i), or Rac1 inhibitor (Cr/Rac1-i) for 48 h, and used for the co-culture with Jurkat cells. The luciferase activities are shown by setting the value of Jurkat alone as 1 (n = 3). *p < 0.05.

We performed co-culture assays to examine how M-Sec contributes to HTLV-1 infection. MT-2 or SLB-1 was used as infected cells, and Jurkat carrying the HTLV-1 LTR promoter-driven luciferase gene was used as the target cells. M-Sec knockdown MT-2 or SLB-1 bulk culture cells were prepared (MSec-KD; S3 Fig ), and M-Sec inhibitor was also used (MSec-i; S3 Fig ). It has been shown that MSec-i reduces the formation of tunneling nanotubes in several cell lines engineered to express M-Sec [ 8 , 22 ] and the production of HIV-1 in the culture of macrophages without affecting podosome formation and phagocytic activity of the cells [ 8 , 23 ]. Control MT-2 or SLB-1 cells were prepared by transducing non-targeting shRNA (Cr; S3 Fig ). We found that M-Sec knockdown in MT-2 or SLB-1 cells, or MSec-i addition to control MT-2 or SLB-1 cells reduced viral infection to target cells ( Fig 4A ), albeit modestly when compared to the anti-envelope (Env) antibody [ 24 ] (Cr/Env Ab).

( A ) The control JET35 (left) or HTLV-1-infected JEX22 cells (right) were left un-stimulated or stimulated with PMA and ionomycin for 16 or 32 h, and analyzed for the expression of M-Sec- (upper) and Tax mRNA (lower) by qRT-PCR. The expression levels are shown by setting the value of un-stimulated cells as 1 (n = 3). *p < 0.05. ( B ) Jurkat cells were nucleofected with the empty vector, or Tax plasmid expressing wild type (WT) or M22 mutant. After 24 h, the cells were analyzed for the expression of M-Sec- (upper) and Tax mRNA (lower) by qRT-PCR. M-Sec expression levels are shown by setting the value of cells nucleofected with the empty vector as 1 (n = 3). Tax expression levels are shown by setting the value of cells nucleofected with the wild type Tax as 1 (n = 3). *p < 0.05. n.s., not significant.

To further analyze M-Sec expression by HTLV-1, we used JEX22, a Jurkat cell-based HTLV-1-infected line that produces HTLV-1 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin [ 20 ]. When stimulated, JEX22 cells expressed Tax and M-Sec ( Fig 3A , right). Such a change was not observed in uninfected control cells (JET35; Fig 3A , left). When expressed in Jurkat cells, Tax induced M-Sec ( Fig 3B ). Interestingly, a Tax mutant M22, which can activate the HTLV-1 LTR promoter but not the NF-κB promoter [ 21 ] ( S2 Fig ), failed to induce M-Sec ( Fig 3B ). Collectively, these results suggest that Tax, which is expressed by the viral plus-strand transcriptional burst, induces M-Sec in CD4 + T cells through potent activation of the NF-κB pathway.

( A ) The indicated T cell lines were analyzed for the expression of M-Sec- and Tax protein by western blotting. α-tubulin blot is the loading control. Monocyte-derived macrophages (MDM) obtained by culturing monocytes from healthy volunteers (#1 and #2) were added as a positive control for M-Sec. ( B ) The indicated T cell lines were co-stained with phalloidin (green, to visualize F-actin) and DAPI (blue). Scale bar: 10 μm. ( C ) KK-1 cells expressing GFP in a Tax-dependent manner were subjected to sorting (Tax - or Tax + fraction) followed by RNA-seq (accession number in NCBI GEO database: GSE108601) [ 18 ]. The RNA-seq data were analyzed for the expression of M-Sec mRNA in those fractions. FPKM, Fragments Per Kilobase of exon per Million mapped reads.

Furthermore, HTLV-1 + Tax + T cell lines (SLB-1 and MT-2) expressed M-Sec protein, the level of which was comparable to that of monocyte-derived macrophages ( Fig 2A ), which are typical M-Sec + cells [ 3 , 8 ]. SLB-1 and MT-2 cells also formed many membrane protrusions ( Fig 2B ), the hallmark activity of M-Sec [ 3 ]. The HTLV-1 - cell line (Jurkat) and HTLV-1 + Tax - cell lines (S1T, ED, and KK-1) did not show clear M-Sec expression and membrane protrusions. Interestingly, KK-1 cells expressed Tax, but the number of Tax + KK-1 cells in the culture was small at any given time because of the sporadic on/off switching of Tax [ 18 ] ( S1 Fig ). The minor Tax + fraction, but not the major Tax - fraction, highly expressed M-Sec ( Fig 2C ). Moreover, in a mouse model of HTLV-1 infection [ 20 ], in which humanized mice were inoculated with MT-2 cells, only Tax high human CD3 + cells expressed M-Sec at a detectable level ( S1 Table , mouse #3 and #4).

( A ) The CD3 + CD4 + CD14 - cells in the live cell gate were sorted from peripheral blood mononuclear cells (PBMCs). The profile of an HTLV-1 carrier is shown as an example. CD14 was used to exclude monocytes, which abundantly express M-Sec [ 3 , 8 ]. ( B ) The sorted CD3 + CD4 + CD14 - cells of HTLV-1 - individuals (HTLV-1−; n = 7) or HTLV-1 carriers (HTLV-1+; n = 7) were analyzed for the expression of M-Sec mRNA by using qRT-PCR, before or after culturing for 18 h. The expression levels are shown by setting the value of un-cultured cells as 1. *p < 0.05. ( C ) The sorted CD3 + CD4 + CD14 - cells of HTLV-1 - individuals (HTLV-1−; #1 and #2) or HTLV-1 carriers (HTLV-1+; #1 and #2) were cultured for the indicated periods and analyzed as in ( B ). The expression levels are shown by setting the value of un-cultured cells as 1.

Recent studies have revealed that the transcription of HTLV-1 provirus is regulated by a unique mechanism⁠. Viral plus-strand transcription is silent in freshly isolated cells of HTLV-1 carriers, but a short-term ex vivo culture induces a spontaneous transcriptional burst of viral genes, including the gene encoding the trans-activating protein Tax [ 17 , 18 ]. In this study, we found that when sorted and cultured ( Fig 1A ), CD4 + T cells of most HTLV-1 carriers tested expressed M-Sec ( Fig 1B ). Such a change was not observed in CD4 + T cells of HTLV-1 - individuals. M-Sec induction in CD4 + T cells of carriers was detected as early as 4 h after the beginning of culture and increased at least up to 48 h ( Fig 1C ). Although we analyzed carriers whose viral load was similar (6.3–13.1%, 9.3 ± 2.6%, n = 7), M-Sec induction was weak or undetected in several carriers ( Fig 1B ). Similar variability was observed in viral transcripts among HTLV-1 + individuals [ 19 ].

Discussion

It has been believed that M-Sec is not related to an infection in T cells because of the lack of their expression under physiological conditions. In this study, we revealed that M-Sec plays a critical role in HTLV-1 infection in CD4+ T cells. Our study suggests that Tax expressed by the viral plus-strand transcriptional burst induces M-Sec through a potent activation of NF-κB pathway, and that M-Sec mediates an efficient cell-to-cell infection of HTLV-1 likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag.

We demonstrated that M-Sec is a Tax-inducible protein. A series of experiments using CD4+ T cells from HTLV-1 carriers (Fig 1), HTLV-1+ cell lines, including SLB-1, MT-2, and KK-1 (Fig 2), and Tax-expressing Jurkat cells (Fig 3) support the idea that Tax translated by the viral plus-strand transcriptional burst induces the expression of M-Sec in CD4+ T cells. Tax-mediated M-Sec induction required activation of the NF-κB pathway (Fig 3B). Tax is known to activate NF-κB [36], while the effects of HIV-1 on NF-κB activation depend on the type of cells or their activation state [37], which explains why M-Sec was not induced in HIV-1-infected Jurkat cells despite active viral replication [8]. The finding that LMP-1, an oncoprotein of Epstein-Barr virus, upregulates M-Sec in nasopharyngeal carcinoma cells through NF-κB activation [5] also supports M-Sec induction in CD4+ T cells by the Tax-NF-κB cascade.

Tax is essential for de novo infection of HTLV-1 as it induces the expression of viral genes, but it simultaneously allows the recognition of infected cells by cytotoxic T lymphocytes [38]. Thus, the transient or intermittent expression of Tax by the viral plus-strand transcriptional burst [17,18] may be a strategy for HTLV-1 to maintain a balance between escape from the immune system and de novo infection [38]. To ensure viral infection within its limited period of expression, Tax may induce cellular proteins including M-Sec, which mediates an efficient transmission of HTLV-1 as demonstrated by our analyses of co-cultures (Figs 4 and 5) and the mouse model (Fig 6).

The ability of M-Sec to enhance migratory activity of infected cells (Fig 7C) can increase the likelihood of encountering target cells, thereby contributing to an efficient cell-to-cell infection. In fact, among Tax-inducible cellular proteins that facilitate contact between HTLV-1-infected cells and target cells [39–41], Gem, a member of the small GTP-binding proteins, not only enhances the migratory activity of infected cells but also mediates an efficient viral infection from infected cells to target cells in a co-culture assay [41]. The ability of M-Sec to enhance the formation of membrane protrusions (Fig 7B) can facilitate contact between the infected cells and target cells. The protrusions of MT-2 or SLB-1 cells (Fig 2B) appear to be smaller in length but larger in number than those of HIV-1-infected macrophages or U87 cells [8,9]. Furthermore, the ability of M-Sec to facilitate the clustering of Gag (Figs 8, S12, and 9) may be beneficial for viral transmission because Gag is the key driver for the formation of viral particles [31–35]. Consistent with this idea, an alanine-scanning mutagenesis analysis of Gag has identified several mutants that not only fail to form puncta but also produce lesser amounts of viral particles when compared to the wild type Gag [34].

M-Sec inhibition in macrophages or knockdown in U87 glioma cells reduced the production of HIV-1 [8,9]. However, such viral reduction became less obvious over time in the culture [9], implying that M-Sec mainly contributes to the initial phase of HIV-1 transmission. In the co-culture of the present study, reduced viral transfer from M-Sec knockdown MT-2 cells to Jurkat cells was observed at 16 h (Fig 4A, upper), but not at 36 h (S13 Fig). In addition, in several cases in the co-culture using CADM1+ T cells of HTLV-1 carriers, the reduced proviral copies by M-Sec inhibition found at 2 days were lost at 4 days (S5 Fig, #4 and #5). Meanwhile, the extent of the reduced proviral copies by M-Sec knockdown in the mouse model found at 4 weeks (Fig 6) appears to be more obvious than that in the co-cultures. Thus, to clarify whether M-Sec mainly contributes to the initial phase of HTLV-1 transmission, detailed time-course analyses will be necessary for the assays involving the mouse model.

M-Sec expression in several cancer cells enhances their invasion/metastasis [5–7] or proliferation [6,7]. In our cultures, M-Sec knockdown or inhibition in MT-2 cells reduced their migration (Fig 7C), but not proliferation (S6 and S8 Figs). When intraperitoneally injected into immunodeficient mice, the numbers of M-Sec knockdown MT-2 cells in the spleen were not different from those of the control MT-2 cells (S7 Fig). These results suggest that M-Sec knockdown does not affect the proliferation or survival of MT-2 cells in mice or the migration from the peritoneal cavity to the spleen. Thus, it is tempting to speculate that a weak intra-tissue migration of M-Sec knockdown MT-2 cells and their reduced membrane protrusions/Gag clustering could explain the reduced viral infection in the mouse model, although further studies including the identification of M-Sec mutants which lack selected functions are necessary to prove this hypothesis.

M-Sec regulates cellular morphology, migration, and membrane protrusions [3–9]. In addition to these well-known abilities, our current study suggests that M-Sec functions as a regulator of HTLV-1 Gag clustering. M-Sec binds phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) [4], but this feature may not explain the newly identified function, since the binding of HTLV-1 Gag to cellular membranes is essentially independent of PI(4,5)P 2 [32,33]. Thus, the strong ability of M-Sec to induce membrane deformation and actin cytoskeleton remodeling [3–6] may explain the regulation of HTLV-1 Gag clustering. Alternatively, cellular protein(s) involved in vesicle trafficking that have been predicted to interact with M-Sec in the STRING database [42,43], or Ral, which is the possible downstream effector of M-Sec [3], may be attributed to the role of M-Sec in HTLV-1 Gag clustering. Unlike HTLV-1 Gag, HIV-1 Gag binds PI(4,5)P 2 [32,33]. Thus, it will be intriguing to test whether M-Sec affects the clustering of HIV-1 Gag.

How different M-Sec functions are related to each other is still unclear. How these functions contributes to HTLV-1 transmission and the extent to which each function contributes remain unexplored. Despite these unresolved questions, the present study revealed the importance of M-Sec for HTLV-1 transmission. M-Sec is a new and useful tool to further clarify the process of cell-to-cell infection of HTLV-1.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010126

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