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The earliest defense against microbial infection is represented by the responses of the innate (or natural) immune system, that also profoundly regulates the adaptive (or acquired) T- and B-cell immune responses.Activation of the innate immune system is primed by microbial invasion in response to conserved structures present in large groups of microorganisms (LPS, peptidoglycan, double-stranded RNA), and is finely tuned by different cell types (including dendritic cells, macrophages, natural killer cells, natural killer T cells, and γδ T cells). In addition, several soluble factors (complement components, defensins, mannose-binding lectins, interferons, cytokines and chemokines) can play a major role in the regulation of both the innate and adaptive immunity.In this review, we will briefly overview the regulation of some cellular subsets of the innate immune system particularly involved in human immunodeficiency virus (HIV) infection and then focus our attention on those cytokines and chemokines whose levels of expression are more profoundly affected by HIV infection and that, conversely, can modulate virus infection and replication. [Copyright &y& Elsevier]

Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl2 or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca2+ signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca2+ signaling through calmodulin-mediated interactions in infected cells. [ABSTRACT FROM AUTHOR]

This study was designed to assess the in vitro effects of morphine on the lymphocytes infected with SIV. CEM x174 cells were cotreated with morphine and simian immunodeficiency virus (SIVmac239). Cells were cultured for 96 h and the effects of morphine on the viability of infected cells were determined. At the concentration of 1 μmol/l, morphine could inhibit the proliferation of CEM x174 cells at the culture of 72 h. The stronger effect was observed in the case of viral infection. During 72 h SIV loading, the cells were accumulated in S phase in all SIV infected groups. The S arrest was observed in every experimental group and statistically different from normal groups (P<0.05). The results from annexin V binding assay showed that SIV infection resulted in a lower proportion of vital cells and higher mortality compared with corresponding control (P<0.01). Morphine failed to induce detectable alteration in the cell cycle profile of viral infected cells. Western blotting showed that the synthesis of intracellular p53 and bax protein was gradually up-regulated in the virus-loading period of 72 h. Naloxone had an apparent additive rather than antagonistic effect on the morphine-associated enhancement of bax expression. The ratio of bax/bcl-2 proteins appeared to tilt the balance toward apoptosis. At 72 h of infection, 1 μmol/l of morphine significantly elevated the level of caspase-3. These results indicated that the alteration in the balance of intracellular apoptotic and anti-apoptotic elements is one of the reasons of accelerated progression of acquired immunodeficiency syndrome (AIDS) by opioids abuse. [Copyright &y& Elsevier]

By the end of 2004, more than 20 HIV-1 vaccine candidates will have entered clinical testing in at least 30 trials worldwide. Almost half of these vaccines include nonstructural HIV-1 gene products. This represents an important innovation in the HIV vaccine field, because until 9 years ago not even preclinical testing in small animal models had been carried out with such immunogens. This review briefly discusses the experimental evidence that provides the rationale for the use of nonstructural HIV-1 gene products as vaccine antigens, and summarizes the current status and the future development of these novel vaccines. [Copyright &y& Elsevier]

Safety-tested modified vaccinia virus Ankara (MVA) has been established as a potent vector system for the development of candidate recombinant vaccines. The versatility of the vector system was recently demonstrated by the rapid production of experimental MVA vaccines for immunization against severe acute respiratory syndrome associated coronavirus. Promising results were also obtained in the delivery of Epstein-Barr virus or human cytomegalovirus antigens and from the clinical testing of MVA vectors for vaccination against immunodeficiency virus, papilloma virus, Plasmodium falciparum or melanoma. Moreover, MVA is considered to be a prime candidate vaccine for safer protection against orthopoxvirus infections. Thus, vector development to challenge dilemmas in vaccinology or immunization against poxvirus biothreat seems possible, yet the right choice should be made for a most beneficial use. [Copyright &y& Elsevier]

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC50<10 nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC50>100 nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors. [Copyright &y& Elsevier]

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: kon,HIV-1=1.3×1015 M−2 s−1 versus kon,SIV=1.1×1010 M−2 s−1. The unfolding rates are similar: koff,HIV-1=1.1×10−5 s−1 versus koff,SIV=5.7×10−4 s−1. Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics. [Copyright &y& Elsevier]

Protein transport into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly evident that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Vpx, a 112 amino acid protein from human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency virus (SIV) is one such protein, which does not have an identifiable canonical NLS and is yet efficiently imported to the nuclear compartment. Here we report that Vpx protein is imported to the nucleus independently of virus-encoded cofactors. When fusions of truncated versions of Vpx with full-length β-galactosidase (β-Gal) were tested, the region from Vpx 61 to 80 was found to be sufficient to mediate the import of the heterologous cytoplasmic protein to the nucleus. Inactivation of Vpx NLS precluded nuclear import of Vpx and reduced virus replication in non-dividing macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral nuclear import were present. Importantly, we identified and characterized a novel type of 20 amino acid transferable nuclear import signal in Vpx that is distinct from other import signals described. In addition, we show that the minimal nuclear targeting domain identified here overlaps with helical domain III (amino acid (aa) 64–82) and the structural integrity of this helical motif is critical for the nuclear import of Vpx. Taken together, these data suggest that Vpx is imported to the nucleus via a novel import pathway that is dependent on its 20 amino acid unique nuclear targeting signal, and that the nuclear import property of Vpx is critical for the optimal virus replication in non-dividing cells such as macrophages. [Copyright &y& Elsevier]

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I β turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5. [Copyright &y& Elsevier]

Background: Current research suggests that human immunodeficiency virus type-1 (HIV-1) virion infectivity factor (Vif) acts during viral assembly in producer cells to ensure infectivity in target cells but the exact mechanism of action has not been defined. Vif interacts with Gag, viral protease and RNA and these interactions are proposed to be important for correct particle assembly and stability of the reverse transcription complex. Objectives: The existence of cells that are either permissive or non-permissive for replication of Vif deficient viruses suggests the involvement of host cellular factors in its function. Current research suggests an association of Vif with the intermediate filament protein, vimentin, and the tyrosine kinase, Hck, but the significance of these associations remains to be defined. More recently HP68, a cellular ATP binding protein, has been shown to be important for capsid formation and an interaction between Vif and HP68 has been shown. Our aim was to further identify host cellular factors involved in Vif function. Study design: We have employed the yeast 2-hybrid system to identify cellular proteins which interact with HIV-1 Vif. Sixteen clones were isolated from a high stringency yeast-2-hybrid screen of a human leucocyte cDNA library with Vif derived from the T-cell tropic HIV-1 strain NL4.3. Of these, 8 clones were confirmed as specifically binding Vif, fully sequenced and identified via GenBank homology searches. Results: Thus far 3 of these clones, spermine/spermidine N1-acetyltransferase, Triad 3 and a novel gene which we have termed ‘novel Vif binding protein’, have been characterised and represent attractive candidates for mediating Vif action during HIV replication. Conclusions: Through identification and characterisation of cellular factors interacting with HIV-1 Vif we hope to unravel the mechanism of action of Vif which may ultimately aid therapeutic design. [Copyright &y& Elsevier]

Pulsed-field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix-loop-helix regulatory Ca2+-binding proteins to characterize the conformational changes associated with Ca2+-saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii ( R h) obtained for apo- and Ca2+-CaM, consistent with previously reported R h data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small-angle X-ray scattering results. Both sets of data demonstrate the well-established collapse of the extended Ca2+-CaM molecule into a globular complex upon peptide binding. The R h of CaM complexes with target peptides from CaM-dependent protein kinase I (CaMKI) and an N-terminal portion of the SIV peptide (SIV-N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV-N shows only a partial collapse. Interestingly, the covalent CaM-cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the R h of calcium and integrin binding protein (CIB), calbrain, and the calcium-binding region from soybean calcium-dependent protein kinase (CDPK) decrease on Ca2+-binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG-diffusion NMR to rapidly and accurately screen for molecular size changes on protein-ligand and protein-protein interactions for this class of proteins. [ABSTRACT FROM AUTHOR]

Bovine immunodeficiency virus (BIV) in Holstein cows was associated with morphologic evidence of lymphoid organ deficiency. Cows were subjected to normal management practices including parturition and lactation without adverse environmental stresses. During the clinical disease process there was marked weight loss and wasting with frequent and severe concurrent infections. Lymphoid follicular hyperplasia and dysplasia in lymph nodes, and hypertrophy and hyperplasia in hemal lymph nodes were characteristics of the lymphoid tissues. Atrophy of lymphoid cell compartments with depletion of lymphocytes and a lymphocytic lymphoid folliculitis were components of the lymphoid system pathology. The nodal tissue lesions resembled those observed in feline, simian, and human lentiviral disease. A functional correlation with immune system deficiency was the development of multiple bacterial infections which failed to resolve after appropriate therapy. The BIV-associated disease syndrome in dairy cows may be useful as a model system for investigation of the pathogenesis of the lymphoid organ changes that occur in humans and animals with lentiviral infection. [ABSTRACT FROM AUTHOR]

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man8 and Man9. Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manα(1-2)Manα (which represents the terminal disaccharide of all three arms of Man9) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manα(1-2)Manα complex revealed that CVN recognizes the stacked conformation of Manα(1-2)Manα through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manα(1-2)Manα(1-2)Man, methyl Manα(1-2)Manα(1-3)Man and methyl Manα(1-2)Manα(1-6)Man with remarkable selectivity, and binds these trisaccharides with KA values ranging from 8.1×103 M−1 to 6.6×106 M−1. Site 2 is less selective in that it binds all three trisaccharides with similar KA values ranging from 1.7 to 3.7(±0.3)×105 M−1, but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display α(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source. [Copyright &y& Elsevier]

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion. [Copyright &y& Elsevier]

Assembly of infectious retroviral particles involves recognition of specific sequences on the viral RNA by the nucleocapsid (NC) domain of the Gag polyprotein, and subsequent stoichiometric binding of the processed NC protein along the entire length of the RNA. NC proteins also act as nucleic acid chaperones. They accelerate nucleic acid hybridization and strand exchange, which may be critical during the initial stages of reverse transcription. In order to better understand these properties, we have studied the nucleic acid helix-destabilizing tm-depressing) and binding activities of HIV-1 NCp7 protein with a variety of substrates, and the real-time kinetics of NC-induced strand exchange. At low ionic strength (0.01 M Na phosphate, pH 7.0) and saturating levels of protein, NCp7 displays moderate helix-destabilizing activity on double-stranded DNA. Saturating levels of NCp7 lowered the tm of a synthetic 28 base-pair 28(+)/28(−) oligonucleotide duplex by about 10 deg. C (51 to 41 °C). The presence of single-stranded calf thymus DNA (equimolar with duplex) eliminated the tm depression, whereas double-stranded calf thymus DNA only altered the tm of the 28-mer duplex by about 2 deg. C. Similar effects were seen with duplexes with single-stranded overhangs or internal single-stranded gaps. Binding experiments utilizing intrinsic tryptophan quenching indicated significant affinity (Kd about 0.1 μM) for both single-stranded and double-stranded forms of the 28-mer in 0.01 M sodium phosphate at 25 °C, although long-chain (calf thymus double-stranded) DNA displayed a much lower affinity. The effects of NCp7 on the kinetics of nucleic acid annealing, strand exchange, and strand displacement were determined by use of oligonucleotides with end-labeled fluorophores serving as donor–acceptor pairs. NCp7 accelerated all these reactions. In the strand exchange reaction, an imperfect duplex, 28(+)/21(−), was reacted with a perfect complement, 28(−). The kinetics of 28(+)/28(−) annealing in this reaction did not conform to a simple bimolecular model, but could be well fit to the sum of two exponential decays. Addition of stoichiometric levels of NCp7 increased the rate constants of both components, and significantly increased the fraction of exchange associated with the rapid process. Increasing levels of 28(−) also increased the rapid fraction, as well as the rapid rate constant. This concentration dependence indicates that, although the kinetic decays appear biexponential, at least one of the steps is bimolecular. Simple annealing reactions, 28(+) with 28(−), could be fit to single-exponential decays, and their magnitudes in the presence of NCp7 were comparable to the rapid step of annealing observed for exchange reactions, suggesting that this step is connected with annealing. Strand dissociation during exchange was monitored by placing the fluorescent acceptor on the 21(−) strand. The results, though complex, suggest that the slow step of exchange is largely associated with the dissociation of the shorter oligonucleotide. Analogous experiments were performed with variants of these oligonucleotides, and the results are in line with the 28(+)/21(−)/28(−) experiments. On the basis of an analysis of the effect of increasing levels of 28(−) on the formation of the perfect 28 bp duplex from the imperfect duplex, we propose that NCp7 forms a ternary complex intermediate with imperfect duplex and 28(−), and suggest several ways by which such an intermediate would facilitate strand exchange. [Copyright &y& Elsevier]

In eukaryotic cells, protein synthesis is regulated by a set of initiation factors (eIF) that are required for recruiting the 40 S ribosomal subunit onto the mRNA molecule. Among these proteins, eIF4GI, which is targeted by picornaviral proteases, makes a bridge between the mRNA cap structure (via eIF4E) and the 40 S ribosome (via eIF3). Recently, internal ribosome entry segment (IRES) elements have been characterized in the genomic RNA of both simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), suggesting that viral expression of these two viruses can be regulated at the translational level. Thus, by analogy with members of the picornavirus family, we have investigated the action of the HIV-1 protease on initiation factors eIF4GI and eIF4GII using cell extracts and the rabbit reticulocyte lysate system. Our results show that eIF4GI, but not eIF4GII, is substrate for HIV-1 protease and this effect can be prevented by a HIV-1 protease inhibitor, palinavir. However, in contrast to picornaviral proteases, the cleavage of eIF4GI by HIV-1 protease occurs at multiple sites and impairs translation of both cap-dependent and IRES-containing RNAs, except for the HCV IRES, which does not require eIF4GI or eIF4GII for activity. [Copyright &y& Elsevier]

The anti-CD4 MAb 5A8 is a potent inhibitor of CD4-mediated infection of HIV-1. CD4 is obligatory for infection with primary HIV-1 isolates. Humanized 5A8 (hu5A8) was constructed to reduce the potential immunogenicity and enhance the in vivo half-life when used in humans. hu5A8 is a molecularly engineered human IgG4 antibody retaining the binding and functional properties of the murine version of 5A8 (mu5A8). This humanized MAb has been shown to be very effective in inhibiting HIV-1 infection of human CD4+ T cells and macrophages in vitro and to reduce viral load in rhesus monkeys chronically infected with simian immunodeficiency virus (SIV). 5A8 was evaluated in a good laboratory practice (GLP)-compliant tissue cross-reactivity study on human (three donors/37 tissues) and rhesus monkey (two donors/37 tissues) tissues. hu5A8 bound to the surface of human T cells and macrophages, but only to T cells from rhesus monkeys. The antibody did not cross react with other tissues. The highly identical staining patterns of hu5A8 in human and rhesus monkey tissues support the use of rhesus monkeys as a preclinical model for humans. In a GLP-compliant safety study in rhesus monkeys, weekly administration of hu5A8 at 5 mg/kg or 25 mg/kg for 8 weeks was shown to be safe and well tolerated in all monkeys. Although hu5A8 induced anti-hu5A8 antibody response in healthy rhesus monkeys, it was not immunogenic in chimpanzees. Together, the results from these preclinical studies support the studies of the anti-HIV-1 effect of hu5A8 in HIV-1 infected individuals. [Copyright &y& Elsevier]

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene.These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene. [Copyright &y& Elsevier]

Virus binding to host cells involves specific interactions between viral (glyco)proteins (GP) and host cell surface receptors (Cr) (protein or sialic acid (SA)). The magnitude of the enthalpy of association changes with temperature according to the change in heat capacity (ΔC p ) on GP/Cr binding, being little affected for avian influenza virus (AIV) haemagglutinin (HA) binding to SA (ΔC p  = 0 kJ/mol/K) but greatly affected for HIV gp120 binding to CD4 receptor (ΔC p  = -5.0 kJ/mol/K). A thermodynamic model developed here predicts that values of ΔC p from 0 to ~-2.0 kJ/mol/K have relatively little impact on the temperature sensitivity of the number of mosquito midgut cells with bound arbovirus, while intermediate values of ΔC p of ~-3.0 kJ/mol/K give a peak binding at a temperature of ~20 °C as observed experimentally for Western equine encephalitis virus. More negative values of ΔC p greatly decrease arbovirus binding at temperatures below ~20 °C. Thus to promote transmission at low temperatures, arboviruses may benefit from ΔC p ~ 0 kJ/mol/K as for HA/SA and it is interesting that bluetongue virus binds to SA in midge midguts. Large negative values of ΔC p as for HIV gp120:CD4 diminish binding at 37 °C. Of greater importance, however, is the decrease in entropy of the whole virus (ΔS a&#95;immob ) on its immobilisation on the host cell surface. ΔS a&#95;immob presents a repulsive force which the enthalpy-driven GP/Cr interactions weakened at higher temperatures struggle to overcome. ΔS a&#95;immob is more negative (less favourable) for larger diameter viruses which therefore show diminished binding at higher temperatures than smaller viruses. It is proposed that small size phenotype through a less negative ΔS a&#95;immob is selected for viruses infecting warmer hosts thus explaining the observation that virion volume decreases with increasing host temperature from 0 °C to 40 °C in the case of dsDNA viruses. Compared to arboviruses which also infect warm-blooded vertebrates, HIV is large at 134 nm diameter and thus would have a large negative ΔS a&#95;immob which would diminish its binding at human body temperature. It is proposed that prior non-specific binding of HIV through attachment factors takes much of the entropy loss for ΔS a&#95;immob so enhancing subsequent specific gp120:CD4 binding at 37 °C. This is consistent with the observation that HIV attachment factors are not essential but augment infection. Antiviral therapies should focus on increasing virion size, for example through binding of zinc oxide nanoparticles to herpes simplex virus, hence making ΔS a&#95;immob more negative, and thus reducing binding affinity at 37 °C., Competing Interests: None declared., (© 2020 Elsevier B.V. All rights reserved.)

Despite significant advances in antiretroviral therapy, increasing drug resistance and toxicities observed among many of the current approved human immunodeficiency virus (HIV) drugs indicate a need for discovery and development of potent and safe antivirals with a novel mechanism of action. Maturation inhibitors (MIs) represent one such new class of HIV therapies. MIs inhibit a late step in the HIV-1 Gag processing cascade, causing defective core condensation and the release of non-infectious virus particles from infected cells, thus blocking the spread of the infection to new cells. Clinical proof-of-concept for the MIs was established with betulinic acid derived bevirimat, the prototype HIV-1 MI. Despite the discontinuation of its further clinical development in 2010 due to a lack of uniform patient response caused by naturally occurring drug resistance Gag polymorphisms, several second-generation MIs with improved activity against viruses exhibiting Gag polymorphism mediated resistance have been recently discovered and are under clinical evaluation in HIV/AID patients. In this review, current understanding of HIV-1 MIs is described and recent progress made toward elucidating the mechanism of action, target identification and development of second-generation MIs is reviewed.

Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV(+) groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2-4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy.