Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5'UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5'UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5'UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.     

Seropositivity is associated with insulin resistance in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register (NOAR): an observational study

Introduction  

Cardiovascular disease (CVD) is the leading cause of death in patients with inflammatory polyarthritis (IP), especially in seropositive disease. In established rheumatoid arthritis (RA), insulin resistance (IR) is increased and associated with CVD. We investigated factors associated with IR in an inception cohort of patients with early IP.     

Protein X of hepatitis B virus: origin and structure similarity with the central domain of DNA glycosylase

Orthohepadnavirus (mammalian hosts) and avihepadnavirus (avian hosts) constitute the family of Hepadnaviridae and differ by their capability and inability for expression of protein X, respectively. Origin and functions of X are unclear. The evolutionary analysis at issue of X indicates that present strains of orthohepadnavirus started to diverge about 25,000 years ago, simultaneously with the onset of avihepadnavirus diversification. These evolutionary events were preceded by a much longer period during which orthohepadnavirus developed a functional protein X while avihepadnavirus evolved without X. An in silico generated 3D-model of orthohepadnaviral X protein displayed considerable similarity to the tertiary structure of DNA glycosylases (key enzymes of base excision DNA repair pathways). Similarity is confined to the central domain of MUG proteins with the typical DNA-binding facilities but without the capability of DNA glycosylase enzymatic activity. The hypothetical translation product of a vestigial X reading frame in the genome of duck hepadnavirus could also been folded into a DNA glycosylase-like 3D-structure. In conclusion, the most recent common ancestor of ortho- and avihepadnavirus carried an X sequence with orthology to the central domain of DNA glycosylase.     

Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1. To better understand fusion inhibitor potency and resistance, we combined virological, computational, and biophysical experiments with comprehensive mutational analyses and tested resistance to T20 and second and third generation inhibitors (T1249 and T2635). We found that most amino acid substitutions caused resistance to the first generation peptide T20. Only charged amino acids caused resistance to T1249, and none caused resistance to T2635. Depending on the drug, we can distinguish four mechanisms of drug resistance: reduced contact, steric obstruction, electrostatic repulsion, and electrostatic attraction. Implications for the design of novel antiviral peptide inhibitors are discussed.The HIV-1 envelope glycoprotein complex (Env),³ a class I viral fusion protein, is responsible for viral attachment to CD4⁺ target T cells and subsequent fusion of viral and cellular membranes resulting in release of the viral core in the cell. Other examples of viruses using class I fusion proteins are Coronaviridae (severe acute respiratory syndrome virus), Paramyxoviridae (Newcastle disease virus, human respiratory syncytial virus, Nipah virus, Hendra virus), and Orthomyxoviridae (influenza virus), some of which cause fatal diseases in humans (1–3). The entry process of these viruses is an attractive target for therapeutic intervention.The functional trimeric Env spike on HIV-1 virions consists of three gp120 and three gp41 molecules that are the products of cleavage of the precursor gp160 by cellular proteases such as furin (4, 5). The gp120 surface subunits are responsible for binding to the cellular receptors, whereas the gp41 subunits anchor the complex in the viral membrane and mediate the fusion of viral and cellular membranes. Env undergoes several conformational changes that culminate in membrane fusion. The gp120 subunit binds the CD4 receptor, resulting in creation and/or exposure of the binding site for a coreceptor, usually CCR5 or CXCR4 (6, 7). Two α-helical leucine zipper-like motifs, heptad repeat 1 (HR1) and heptad repeat 2 (HR2), located in the extracellular part of gp41, play a major role in the following conformational changes. Binding of the receptors to gp120 induces formation of the pre-hairpin intermediate of gp41 in which HR1 is exposed and the N-terminal fusion peptide is inserted into the target cell membrane (1, 8–12). Subsequently, three HR1 and three HR2 domains assemble into a highly stable six-helix bundle structure that juxtaposes the viral and cellular membranes for the membrane merger. Other viruses with class I viral fusion proteins use similar HR1-HR2-mediated membrane fusion for target cell entry.Peptides based on the HR domains of class I viral fusion proteins have proven to be efficient inhibitors of virus entry for a broad range of viruses (13–17). The HIV-1 fusion inhibitor T20 (enfuvirtide (Fuzeon)) has been approved for clinical use. T20 mimics HR2 and can bind to HR1, thereby preventing the formation of the six-helix bundle (Fig. 1) (18–21). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared with the first-generation peptide T20 (22–25). Recently, a series of more potent third-generation fusion inhibitors were designed (26, 27). These include T2635, which has an improved helical structure that increases stability and activity against both wild type (WT) HIV-1 and fusion inhibitor resistant variants.Open in a separate windowFIGURE 1.Schematic of the gp41 ectodomain. HR1 and HR2 are represented as cylinders, and position 38 in HR1 is indicated. Residues Gln-142, Asn-145, Glu-146, and Leu-149, which interact with residue 38, are underlined in the HR2 sequence. HR2-based peptide fusion inhibitors are shown underneath. Mutations introduced in T1249^mut and T2635^mut are bold and underlined. Numbering is based on the sequence of HXB2 gp41.Both the in vitro and in vivo selection of resistance has been described for T20 (28–33) and T1249 (23, 34–36). Resistance is often caused by mutations in the HR1 binding site of the fusion inhibitor. In particular, substitutions at positions 36 (G36D/M/S), 38 (V38A/W/M/E), and 43 (N43D/K) of gp41 can cause resistance. Strikingly, substitutions at position 38 can cause resistance to both T20 and T1249, but distinct amino acid substitutions are required. At position 38 only charged amino acids (V38E/R/K) cause resistance to T1249 (35). Surprisingly, none of the known T20 and T1249 resistance mutations at position 38 affect the susceptibility to the third generation inhibitor T2635.We hypothesized that the use of HIV-1 as a model system could provide a more detailed understanding of resistance to fusion inhibitors. We, therefore, analyzed the effect of all 20 amino acids at resistance hotspot 38 on Env function, viral fitness, biochemical properties of gp41, and resistance to the fusion inhibitors. From the results we can propose four resistance mechanisms that differ in the way the drug-target interaction is affected at the molecular level. Furthermore, we can deduce general principles on the mechanisms of resistance against fusion inhibitors and the requirements for effective antiviral drugs.     

Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.