Human T-cell leukemia virus Type 1 (HTLV-1), the only human retrovirus causing cancer

HTLV-1 is a highly oncogenic yet neglected retrovirus, which primarily infects white blood cells (CD4+ T-cells) in vivo and causes incurable diseases like Adult T-cell leukemia (ATL) or inflammatory maladies like HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP) after lifelong viral persistence. Worldwide, at least 5-10 million people are HTLV-1-infected and most of them are unaware of their infection. Endemic regions are located in Japan, Central Australia, Melanesia, South America, the Caribbean, and the Middle East. The virus is transmitted via cell-containing body fluids such as blood products, semen and breast-milk, which constitutes the major route of mother-to-child transmission. After infection of CD4+ T-cells, HTLV-1 is reversely transcribed and integrates into the host cell genome. Despite the high number of people worldwide living with a persistent HTLV-1 infection, there is still no efficient prevention strategy, vaccine or cure for HTLV-1 and its associated diseases.

Our main research areas are:

Transmission of retroviruses by breast milk and development of prevention strategies: Milk-Transmission of Viruses (Milk-TV)

We are happy to announce that the Federal Ministry of Education and Research (BMBF) supports our work by funding a Junior Research Group in Infection Research. Within the project Milk-TV, which is short for “Milk Transmission of Viruses”, we seek to explore early events of HTLV-1 transmission and to develop prevention strategies to fight HTLV-1-infections. Briefly, breastfeeding is recommended by the World Health Organization for at least 6 months and up to 2 years of age, and it is proven that breast milk protects against several diseases and viral infections. Intriguingly, few viruses are preferentially transmitted via breastfeeding, including HTLV-1 (Kemeter et al., 2023). Risk of transmission increases with the duration of breastfeeding; however, abstinence from breastfeeding is not an option in resource-limited settings or underrepresented areas or populations. Despite significant progress in understanding details of cell-to-cell transmission, it is still unclear which cells in which organs get infected via the oral route, how these cells get infected, how breast milk affects this route of infection and how to inhibit oral transmission despite breastfeeding, which is an urgent need especially in underrepresented areas of the world. Thus, we strive within this project to address these questions experimentally with the help of different tissue culture model systems and the development of strategies to block HTLV-1 transmission. Overall, our central aim is to uncover prevention strategies that might ultimately allow infants to benefit from breastfeeding while reducing the risk of HTLV-1 transmission (Millen and Thoma-Kress, 2022).

Molecular insights into HTLV-1-transmission and cell-cell-communication

Transmission of HTLV-1 to other cells is strictly dependent on cell-cell contacts, and viral particles are transferred to other cells after polarized budding at a tight and confined cell-cell contact, the so-called virological synapse. We found that the actin-bundling protein Fascin, which is transcriptionally-induced by the viral Tax protein, is crucial for HTLV-1 cell-to-cell transmission (Gross et al., 2016; Mohr et al., 2015; Heym et al., 2022). Repression of Fascin not only impaired release of viral particles but also cell-to-cell transmission in different model systems. Imaging revealed that Fascin’s localization at tight cell-cell contacts is accompanied by polarization of the Gag proteins, suggesting that Fascin directly affects the distribution of Gag to budding sites and, therefore, viral transmission (Gross et al., 2016; Wiesmann et al., 2016).

HTLV-1 transmission may also occur via viral biofilms at virological synapses, an agglomeration of viruses that are packaged on top of the infected cells in a microbial biofilm-like structure. Our work shed further light on the composition of theses viral biofilms. Based on transcriptomics and data mining, we found that the structural protein Collagen IV (COL4A1/ COL4A2) is transcriptionally regulated by Tax and a component of viral biofilms, with COL4A2 appearing to impact virus transmission. Thus, our study provided first links between Tax’s activity and formation of the viral biofilm by hijacking COL4 protein functions (Millen et al., 2019).

HTLV-1 is not only transmitted at tight cell-cell contacts but transmission may also occur via long-distance connections, which are induced by the small accessory viral protein p8. This protein is generated by proteolytic cleavage from the precursor p12 encoded on open reading frame I of the HTLV-1 genome. p8 induces cellular protrusions and is transferred to other cells to foster HTLV-1 cell-to-cell transmission. To get molecular insights into p8 transfer and HTLV-1-transmission, we made use of bioinformatics in combination with proteomics. Our studies identified novel interaction partners of p8, amongst them, vasodilator stimulated phosphoprotein (VASP). After having developed a novel flow cytometry-based assay which allows rapid and automatic quantitation of p8 transfer to target cells (Donhauser et al., 2018), we could show that knockout of VASP delays the transfer of p8 between cells, most likely by impairing recruitment of p8 to the plasma membrane (Donhauser et al., 2020). Importantly, this study revealed that VASP is also important for HTLV-1 cell-to-cell-transmission (Figure 1).

Figure 1. p8 is transferred to target cells via VASP-containing protrusions. Immunofluorescence staining of co-cultures of Jurkat T-cells expressing FLAG-VASP (blue) and p8-HA (red) with Jurkat T-cells pre-stained with the live cell marker Calcein (green). White arrow: p8 co-localizing with VASP in a protrusion; black arrow: p8 in co-cultured target Jurkat T-cell. Taken from: Donhauser N, Socher E, Millen S, Heym S, Sticht H, Thoma-Kress AK (2020) Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8. PLoS Pathog 16(9): e1008879. 

Regulation of HTLV-1 transcription

The viral transactivator Tax stimulates viral replication by recruiting host cell factors like positive transcription elongation factor b (p-TEFb) to the viral promoter and by enhancing mitotic expansion of infected cells. Since viral gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, new therapeutic strategies aim to transiently activate viral gene expression to shift the equilibrium in favor of an enhanced CTL response to enhance immunogenicity of HTLV-1 Tax, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Thus far, the composition of the protein complex guiding viral gene expression is only partially understood, and systematic analyses and comparisons of compounds affecting viral transcription are lacking. In earlier work, we could identify strong and specific upregulation of the transcription elongation factor ELL2 in HTLV-1-infected cells (Mann et al., 2014). We found that ELL2 strongly enhances Tax-mediated transactivation of the HTLV-1 promoter, and that ELL2 and Tax are part of a common protein complex. However, the detailed composition of this complex and its impact on viral reactivation are unclear. We recently uncovered important domains within Tax and ELL2 which are crucial for Tax:ELL2 complex formation (Kohrt et al., 2021). In one of our recent projects within the DFG funded GRK2504 , we aim to discover strategies to interfere with HTLV-1 transcription and, thus, to enhance immunogenicity of HTLV-1 (Schnell et al., 2021; 2022).

Impact of viral proteins on the cellular transcriptome

Our lab has a long-lasting interest in how viral proteins modulate the cellular transcriptome. In earlier studies, we focused on signaling induced by the viral Tax-1 protein and could identify novel cellular target genes of Tax that are crucial for virus transmission, cellular proliferation, and invasive migration (Kress et al., 2011; Gross et al., 2016; Millen et al., 2019). In a current project funded by the Interdisciplinary Center for Clinical Research (IZKF) at Universitätsklinikum Erlangen and FAU Erlangen-Nürnberg, we use state-of-the-art sequencing techniques to analyze the impact of viral proteins on early events in the cellular transcriptome upon infection of primary cells. These studies will help to provide a better understanding of how HTLV-1 impacts the transcriptomes of the target cells upon de novo infection, thus, providing novel insights into HTLV-1’s capacity to perturbate T-cell signaling.

Tumor induction and interplay with the tumor microenvironment

It has been known for years that constitutive activation of the classical and alternative NF-κB signaling pathways by the viral oncoprotein Tax is a hallmark of HTLV-1-driven cancer. NF-κB-deficient Tax transgenic mice lack the induction of ATL-associated aggressive skin diseases. Further, animal studies therapeutically targeting NF-κB slow down and reduce tumor growth in ATL-like diseases. Although there are different reports whether NF-κB is critical for initiating cellular transformation, there is a strong connection between Tax, NF-κB, tumor formation, and maintenance. We could recently show that activation of NF-κB signaling specifically enhances the abundance of Tax protein, but not of Tax transcripts (Millen et al., 2020). This led to the identification of a positive feedback loop between Tax and NF-κB activity, which results in enhanced protein expression of Tax and might thus serve as a novel therapeutic target to interfere with Tax-driven transformation. To study the impact of NF-κB activity on Tax expression in primary T-cells, we optimized in collaboration with Dr. Ciminale (Padova, Italy) a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA (Manicone et al., 2017). Our results showed that the RNA transfection technique combines high transfection efficiencies with low toxicity in primary T-cells. Together, these findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo. In a collaboration with Dr. Calabro (Padova, Italy), we seek to elucidate the interplay between HTLV-1-infected T-cells and the tumor microenvironment making use of a mouse model of Adult T-cell leukemia.

Figure 2. Schematic working model of a potential positive feedback loop between Tax and NF-κB activity. Components of the NF-κB signaling cascade with an experimentally shown impact on Tax protein expression are colored in red. Taken from: Millen S, Meretuk L, Göttlicher T, Schmitt S, Fleckenstein B, Thoma-Kress AK (2020). A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-κB activity in T-cells. Retrovirology. 17(1):30. doi: 10.1186/s12977-020-00538-w.