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1. Cell size dependent migration of T-cells latently infected with HIV

   Bohn-Wippert, K. ; Dar, R.D. ( March 2020 , Journal of life sciences)

   null (Ed.)

   Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent. In cell migration assays, migrating cells are consistently larger than non-migrating cells. This effect is drug-treatment independent. The cell size dependent motility observed in a previously generated Jurkat latency model correlates with the motility of primary human CD4+ T-cells containing a modified HIV-1 full-length construct JLatd2GFP. In addition, large migrating T-cells, latently infected with HIV, show a slightly decreased rate of reactivation from latency. these results demonstrate that HIV reactivation is cell migration-dependent, where host cell size acts as a catalyst for altered migration velocity. We believe that host cell size controlled migration uncovers an additional mechanism of cellular controlled viral fate determination important for virus dissemination and reactivation from latency. This observation may provide more insights into viral-host interactions regulating cell migration and reactivation from latency and helps in the design and implementation of novel therapeutic strategies. 

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2. Differential recognition of canonical NF-κB dimers by Importin α3

   https://doi.org/10.1038/s41467-022-28846-z  

   Florio, Tyler J. ; Lokareddy, Ravi K. ; Yeggoni, Daniel P. ; Sankhala, Rajeshwer S. ; Ott, Connor A. ; Gillilan, Richard E. ; Cingolani, Gino ( March 2022 , Nature Communications)

   Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin β. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling.

    

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3. Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses

   https://doi.org/10.1038/s41592-021-01328-8  

   Albanese, Manuel ; Ruhle, Adrian ; Mittermaier, Jennifer ; Mejías-Pérez, Ernesto ; Gapp, Madeleine ; Linder, Andreas ; Schmacke, Niklas A. ; Hofmann, Katharina ; Hennrich, Alexandru A. ; Levy, David N. ; et al ( December 2021 , Nature Methods)

   Abstract CD4 + T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4 + T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4 + T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4 + T cells. 

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4. Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation

   https://doi.org/10.1093/nar/gkab874  

   Chen, Wei ; Lu, Wei ; Wolynes, Peter G ; Komives, Elizabeth A ( October 2021 , Nucleic Acids Research)

   Abstract Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors. 

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5. HIV efficiently infects T cells from the endometrium and remodels them to promote systemic viral spread

   https://doi.org/10.7554/eLife.55487  

   Ma, Tongcui ; Luo, Xiaoyu ; George, Ashley F ; Mukherjee, Gourab ; Sen, Nandini ; Spitzer, Trimble L ; Giudice, Linda C ; Greene, Warner C ; Roan, Nadia R ( May 2020 , eLife)

   The female reproductive tract (FRT) is the most common site of infection during HIV transmission to women, but viral remodeling complicates characterization of cells targeted for infection. Here, we report extensive phenotypic analyses of HIV-infected endometrial cells by CyTOF, and use a ‘nearest neighbor’ bioinformatics approach to trace cells to their original pre-infection phenotypes. Like in blood, HIV preferentially targets memory CD4+ T cells in the endometrium, but these cells exhibit unique phenotypes and sustain much higher levels of infection. Genital cell remodeling by HIV includes downregulating TCR complex components and modulating chemokine receptor expression to promote dissemination of infected cells to lymphoid follicles. HIV also upregulates the anti-apoptotic protein BIRC5, which when blocked promotes death of infected endometrial cells. These results suggest that HIV remodels genital T cells to prolong viability and promote viral dissemination and that interfering with these processes might reduce the likelihood of systemic viral spread. 

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