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.

SignificanceAlthough 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.

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular anti-viral effectors, called restriction or resistance factors. The host anti-viral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production, and cytidine-to-uridine driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3'-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires A3G’s specific interaction with reverse transcriptase (RT) itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between RT, A3G and the cellular DNA repair machinery, and identify the suppression of HIV-1 RT by a directly interacting host protein as a new cell-mediated anti-viral mechanism.

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.

Interferons (IFNs) mediate cellular defence against viral pathogens by upregulation of interferon-stimulated genes (ISGs) whose products interact with viral components or alter cellular physiology to suppress viral replication ( 1 – 3 ). Among the ISGs that can inhibit influenza A virus (IAV) ( 4 ) are the myxovirus resistance 1 (MX1) GTPase ( 5 ) and IFN-induced transmembrane protein 3 (IFITM3) ( 6 , 7 ). Here we use ectopic expression and gene knock-out to demonstrate that the IFN-inducible 219 amino acid short isoform of human nuclear receptor coactivator 7 (NCOA7) is an inhibitor of IAV as well as other viruses that enter the cell by endocytosis, including hepatitis C virus (HCV). NCOA7 interacts with the vacuolar H + -ATPase (V-ATPase) and its expression promotes cytoplasmic vesicle acidification, lysosomal protease activity and the degradation of endocytosed antigen. Step-wise dissection of the IAV entry pathway demonstrates that NCOA7 inhibits fusion of the viral and endosomal membranes and subsequent nuclear translocation of viral ribonucleoproteins (vRNPs). NCOA7, therefore, provides a mechanism for immune regulation of endo-lysosomal physiology that not only suppresses viral entry into the cytosol from this compartment but may also regulate other V-ATPase-associated cellular processes such as physiological adjustments to nutritional status, or the maturation and function of antigen presenting cells.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4+ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1.

؉ T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG) and was recently identified as a late postentry suppressor of human immunodeficiency virus type 1 (HIV-1) infection, inhibiting the nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2-mediated inhibition, the precise mechanism of antiviral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses, including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related and share similar domain architectures and structures. However, MX2 possesses an extended N terminus that is essential for antiviral function and confers anti-HIV-1 activity on MX1 [MX1(NMX2)]. Higher-order oligomerization is required for the antiviral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking and coimmunoprecipitation. We show that while monomeric MX2 and MX1(NMX2) mutants are not antiviral, higher-order oligomerization does not appear to be required for full antiviral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for the effective inhibition of HIV-1.IMPORTANCE Interferon plays an important role in the control of virus replication during acute infection in vivo. Recently, cultured cell experiments identified human MX2 as a key effector in the interferon-mediated postentry block to HIV-1 infection. MX2 is a member of a family of large dynamin-like GTPases that includes MX1/MXA, a closely related interferon-inducible inhibitor of several viruses, including FLUAV, but not HIV-1. MX GTPases form higher-order oligomeric structures, and the oligomerization of MX1 is required for inhibitory activity against many of its viral targets. Through structure-function studies, we report that monomeric mutants of MX2 do not inhibit HIV-1. However, in contrast to MX1, oligomerization beyond dimer assembly does not seem to be required for the antiviral activity of MX2, implying that fundamental differences exist between the antiviral mechanisms employed by these closely related proteins.