The long-held view that gamma delta (γδ) T cells in mice and humans are fundamentally dissimilar, as are γδ cells in blood and peripheral tissues, has been challenged by emerging evidence of the cells’ regulation by butyrophilin (BTN) and butyrophilin-like (BTNL) molecules. Thus, murine Btnl1 and the related gene, Skint1, mediate T cell receptor (TCR)-dependent selection of murine intraepithelial γδ T cell repertoires in gut and skin, respectively; BTNL3 and BTNL8 are TCR-dependent regulators of human gut γδ cells; and BTN3A1 is essential for TCR-dependent activation of human peripheral blood Vγ9Vδ2⁺ T cells. However, some observations concerning BTN/Btnl molecules continue to question the extent of mechanistic conservation. In particular, murine and human gut γδ cell regulation depends on pairings of Btnl1 and Btnl6 and BTNL3 and BTNL8, respectively, whereas blood γδ cells are reported to be regulated by BTN3A1 independent of other BTNs. Addressing this paradox, we show that BTN3A2 regulates the subcellular localization of BTN3A1, including functionally important associations with the endoplasmic reticulum (ER), and is specifically required for optimal BTN3A1-mediated activation of Vγ9Vδ2⁺ T cells. Evidence that BTNL3/BTNL8 and Btnl1/Btnl6 likewise associate with the ER reinforces the prospect of broadly conserved mechanisms underpinning the selection and activation of γδ cells in mice and humans, and in blood and extralymphoid sites. Significance Although gamma delta (γδ) T cells compose an evolutionarily conserved third lineage of diversified lymphocytes, alongside αβ T cells and B cells, they can seem overtly different across species and tissues. Thus, human blood γδ cells show butyrophilin (BTN)3A1-dependent responses to metabolites (“phosphoantigens”) not seen by rodent γδ cells, whereas some rodent, γδ-rich compartments, notably in the skin, lack obvious human counterparts. Recently, however, mouse and human intraepithelial gut γδ cells were found to be regulated by pairings of BTN-like genes. This study now shows that BTN3A1 also functions as a pairing, with its subcellular trafficking and optimal activity both regulated by BTN3A2. Hence, seemingly diverse γδ cell biologies across species and tissues are underpinned by conserved mechanisms.

Human immunodeficiency virus (HIV)-based lentiviral vectors (LVs) hold immense promise for gene delivery applications because of their relatively large packaging capacity and their ability to infect a range of cell types. The genome of HIV non-specifically integrates into the host genome, and this promotes efficient, stable transgene expression in dividing cells. However, integration can also be problematic because of variations in gene expression among cells, possible gene silencing and, most importantly, insertional mutagenesis which can lead to undesirable effects such as malignant transformation. In order to alleviate these problems, we have developed a range of non-integrating LVs (NILVs) by introducing point mutations into the catalytic site, chromosome binding site, and viral DNA binding site of the viral integrase (IN). In addition, we have mutated the IN attachment (att) sites within the HIV long terminal repeats (LTRs). All of the vectors produced show efficient reverse transcription and transgene expression in dividing cells and prolonged expression in non-dividing myotubes. Finally, we show that NILV can be used for achieving highly effective gene transfer and expression in muscle in vivo.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences. APOBEC3 proteins are cell-encoded restriction factors that counteract infections, particularly by retroviruses such as HIV-1, and retrotransposons. When packaged into HIV-1 particles, APOBEC3G and APOBEC3F both inhibit reverse transcription and induce destructive hypermutation in viral DNA. The mechanism of APOBEC3 virion packaging awaits elucidation, though a dependency on RNA binding has been established. Here, we employed a cross-linking and next generation sequencing approach to determine which RNAs are bound to A3G and A3F in HIV-1 infected cells. We show that both proteins bind to multiple different RNAs, including viral RNA as well as cellular coding and non-coding RNAs, with relatively little evidence of selectivity. We then developed a complementation assay to address the diversity of RNAs that can act as substrates for A3G/F virion packaging. Consistent with the RNA binding profiles, many RNAs can promote packaging provided that those RNAs are, themselves, packaged. These observations suggest that APOBEC3 packaging lacks selectivity and is driven simply by the non-specific RNA binding capabilities of these proteins. We speculate that this model accounts for the broad range of retro-elements that are susceptible to repression by individual APOBEC3 proteins, and also that such substrates cannot escape APOBEC3-mediated inhibition through sequence variation.