Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes. However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection. While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored. Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation. Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression. CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics. Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation. In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression. We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.

Activation-neutral gene editing of tonsillar CD4 T cells for functional studies in human ex vivo tonsil cultures.

The molecular and immunological properties of tissue-resident resting CD4 T cells are understudied due to the lack of suitable gene editing methods. Here, we describe the ex vivo culture and gene editing methodology ediTONSIL for CD4 T cells from human tonsils. Optimized CRISPR-Cas9 RNP nucleofection results in knockout efficacies of over 90% without requiring exogenous activation. Editing can be performed on multiple cell types in bulk cultures or on isolated CD4 T cells that can be labeled and reintroduced into their tissue environment. Importantly, CD4 T cells maintain their tissue-specific properties such as viability, activation state, or immunocompetence following reassembly into lymphoid aggregates. This highly efficient and versatile gene editing workflow for tonsillar CD4 T cells enables the dissection of molecular mechanisms in ex vivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types.

ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells.

CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.