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Background: Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis. The SU or surface subunit, gp51, of its envelope glycoprotein is involved in receptor recognition and virion attachment. It contains the major neutralizing and CD4+ and CD8+ T cell epitopes found in naturally infected animals. In this study, we aimed to determine global variation and conservation within gp51 in the context of developing an effective global BLV vaccine. Results: A total of 256 sequences extracted from the NCBI database and collected in different parts of the world, were studied to identify conserved segments along the env gene sequences that encode the gp51 protein. Using the MEME server and the conserved DNA Region module for analysis within DnaSP, we identified six conserved segments, referred to as A-F, and five semi-conserved segments, referred to as G-K. The amino acid conservation ranged from 98.8 to 99.8% in conserved segments A to F, while segments G to K had 89.6-95.2% conserved amino acid sequence. Selection analysis of individual segments revealed that residues of conserved segments had undergone purifying selection, whereas, particular residues in the semi-conserved segments are currently undergoing positive selection, specifically at amino acid positions 48 in segment K, 74 in segment G, 82 in segment I, 133 and 142 in segment J, and residue 291 in segment H. Each of the codons for these six residues contain the most highly variable nucleotides within their respective semi-conserved segments. Conclusions: The data described here show that the consensus amino acid sequence constitutes a strong candidate from which a global vaccine can be derived for use in countries where eradication by culling is not economically feasible. The most conserved segments overlap with amino acids in known immunodeterminants, specifically in epitopes D-D', E-E', CD8+ T-cell epitopes, neutralizing domain 1 and CD4+ T-cell epitopes. Two of the segments reported here represent unique segments that do not overlap with previously identified antigenic determinants. We propose that evidence of positive selection in some residues of the semi-conserved segments suggests that their variation is involved in viral strategy to escape immune surveillance of the host. [ABSTRACT FROM AUTHOR]

Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production. [ABSTRACT FROM AUTHOR]

The HIVs have evolved by selecting means to hijack numerous host cellular factors. HIVs exploit the transcription factor NF-κB to ensure efficient LTR-driven gene transcription. However, NF-κB is primarily known to act as a key regulator of the proinflammatory and antiviral responses. Interestingly, retroviruses activate NF-κB during early stages of infection to initiate proviral genome expression while suppressing it at later stages to restrain expression of antiviral genes. During HIV-1 infection, diverse viral proteins such as Env, Nef and Vpr have been proposed to activate NF-κB activity, whereas Vpu has been shown to inhibit NF-κB activation. It is still unclear how HIV-2 regulates NF-κB signaling pathway during its replication cycle. Here we confirm that human BST-2 and HIV-1 Env proteins can trigger potent activation of NF-κB. Importantly, we demonstrate for the first time that the HIV-2 Env induces NF-κB activation in HEΚ293T cells. Furthermore, the anti-BST-2 activity of the HIV-2 Env is not sufficient to completely inhibit NF-κB activity. [ABSTRACT FROM AUTHOR]

The epidemiological link in the hypervariable env gene between viruses infecting HIV-positive hemophiliacs (HPs) and plasma donors was not studied. We determined full-length env gene sequences in 20 HPs, 3 plasma donors whose plasma was used for domestic clotting factor (DCF) production, and 54 local controls (LCs). Env genes from viruses in frozen stored sera obtained 1-3 years after diagnosis and from samples collected several years after infection were amplified via RT-PCR and subjected to direct sequencing. Phylogenetic analysis indicated that all sequences were subtype B, including 133 sequences from 77 cases (20 HPs, 3 plasma donors, and 54 LCs) belonging to the Korean subclade B (KSB) and 6 sequences from 5 cases that did not belong to the KSB. Env gene sequences from donors O and P and those of the 20 HPs comprised 2 subclusters within the KSB, although phylogenetic analysis did not support significant bootstrap values. In contrast, signature pattern analysis indicated signature nucleotides at 43 positions between the HPs and LCs ( P < 0.05). In particular, specific signature nucleotides at 4 positions were fully conserved in the HPs, but not in the LCs ( P < 0.0001). Furthermore, there were 26 signature residues within the KSB and were distinct from the worldwide consensus for subtype B. In conclusion, signature pattern analysis for the hypervariable env gene revealed an epidemiological link that the 20 HPs in this study had been infected with viruses from the DCF used for treatment, consistent with our previous finding. [ABSTRACT FROM AUTHOR]

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies. [ABSTRACT FROM AUTHOR]

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1. [ABSTRACT FROM AUTHOR]

Human endogenous retroviruses (HERV) env proteins have been recently reported to be significantly up-regulated in certain cancers. Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy.

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.

Extensive research on generating an efficient HIV vaccine is ongoing. A major aim of HIV vaccines is the induction of long-lasting, broadly neutralizing antibodies (bnAbs) that can confer sterile immunity for a prolonged period of time. Several strategies have been explored to reach this goal, i.e. protein immunization, DNA, or viral vectors, or a combination thereof. In this review, we give an overview of approaches using viral vectors for the induction of HIV-specific bnAbs. Many pre-clinical studies were performed using various replication-competent and -incompetent vectors. Amongst them, poxviral and adenoviral vectors were the most prevalent ones. In many studies, viral vectors were combined with a DNA prime or a protein boost. However, neutralizing antibodies were mainly induced against the homologous HIV-1 vaccine strain or tier 1 viruses, and in rare cases, against tier 2 viruses, indicating the need for improved antigens and vaccination strategies. Furthermore, we also review next generation Env antigens that are currently being used in protein vaccination approaches and point out how they could be utilized in viral vectors.

The HIV-1 entry-route is a matter of ongoing controversy, and there is evidence for fusion either at the cell surface or from within endosomes. A recent report demonstrated that isoform 4 of nuclear receptor coactivator 7 (NCOA7iso4) interacts with endolysosomal vacuolar-type H+-ATPase (V-ATPase), increasing lytic activity and thereby severely affecting the entry of vesicular stomatitis virus glycoprotein (VSV-G)-mediated, but not HIV-Env-mediated, entry and infection. As basal expression of NCOA7iso4 is low in the absence of type-1 interferons, its overexpression is a novel tool to study viral entry.

Autophagy is a lysosomal-dependent degradative process essential for maintaining cellular homeostasis, and is a key player in innate and adaptive immune responses to intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). In HIV-1 target cells, autophagy mechanisms can (i) selectively direct viral proteins and viruses for degradation; (ii) participate in the processing and presentation of viral-derived antigens through major histocompatibility complexes; and (iii) contribute to interferon production in response to HIV-1 infection. As a consequence, HIV-1 has evolved different strategies to finely regulate the autophagy pathway to favor its replication and dissemination. HIV-1 notably encodes accessory genes encoding Tat, Nef and Vpu proteins, which are able to perturb and hijack canonical and non-canonical autophagy mechanisms. This review outlines the current knowledge on the complex interplay between autophagy and HIV-1 replication cycle, providing an overview of the autophagy-mediated molecular processes deployed both by infected cells to combat the virus and by HIV-1 to evade antiviral response. [ABSTRACT FROM AUTHOR]

Introduction: Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens. Areas covered: We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike. Expert commentary: Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates. [ABSTRACT FROM PUBLISHER]

Collective evidence supporting a role of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression emerged in the last few years. Non-neutralizing antibodies (nnAbs) recognizing conserved CD4-induced epitopes on Env and able to mediate potent ADCC against HIV-1-infected cells exposing Env in its CD4-bound conformation have been shown to be present in some RV144 vaccinees and most HIV-1-infected individuals. HIV-1 evolved sophisticated strategies to decrease exposure of this Env conformation by downregulating CD4 and by limiting the overall amount of cell-surface Env. In this review, we will summarize our contribution to this rapidly evolving field, discuss how structural properties of HIV-1 Env might have contributed to the modest efficacy of the RV144 trial and how we recently used this knowledge to develop new strategies aimed at sensitizing HIV-1-infected cells to ADCC mediated by easy to elicit nnAbs. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. [ABSTRACT FROM AUTHOR]

It has been known for more than 30 years that HIV-1 infection drives a very potent B cell response resulting in the production of anti-HIV-1 antibodies targeting several viral proteins, particularly its envelope glycoproteins (Env). Env epitopes are exposed on the surfaces of viral particles and infected cells where they are targets of potentially protective antibodies. These antibodies can interdict infection by neutralization and there is strong evidence suggesting that Fc-mediated effector function can also contribute to protection. Current evidence suggests that Fc-mediated effector function plays a role in protection against infection by broadly neutralizing antibodies and it might be important for protection by non-neutralizing antibodies. Fc-mediated effector function includes diverse mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation, antibody-dependent cellular phagocytosis, antibody-dependent cell-mediated virus inhibition, antibody-mediated trancytosis inhibition, and antibody-mediated virus opsonization. All these functions could be beneficial in fighting viral infections, including HIV-1. In this perspective, we discuss the latest developments in ADCC research discussed at the HIVR4P satellite session on non-neutralizing antibodies, with emphasis on the mechanisms of ADCC resistance used by HIV-1, the structural basis of epitopes recognized by antibodies that mediate ADCC, natural killer-cell education and ADCC, and murine models to study ADCC against HIV-1. [ABSTRACT FROM AUTHOR]

Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibodydependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV- 1-infected cells to ADCC mediated by HIV-positive (HIVα) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIVα sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals. IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIVα sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpumediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells. [ABSTRACT FROM AUTHOR]

Recent studies have linked antibody Fc-mediated effector functions with control of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in RV144 vaccine trial participants correlated inversely with HIV-1 acquisition risk. These antibodies were recently found to recognize epitopes on the HIV-1 envelope (Env) glycoprotein exposed upon Env-CD4 binding. Accordingly, small-molecule CD4 mimetics (CD4mc) that induce Env to sample the CD4-bound conformation were shown to sensitize HIV-1-infected cells to ADCC mediated by sera from HIV-1-infected individuals. However, it remains unknown whether antibodies elicited through immunization can also mediate CD4mc-induced ADCC. In this study, we tested the capacity of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from Env-vaccinated nonhuman primates using a FACS-based ADCC assay. In parallel, we evaluated the ability of CD4mc to sensitize HIV-1 viral particles to neutralization by sera from these immunized animals. We found that the vaccine-induced antibodies were able to mediate ADCC and viral neutralization in the presence, but not the absence, of CD4mc. Thus, CD4mc are capable of sensitizing HIV-1-infected cells to ADCC and infectious viral particles to neutralization by easy-to-elicit antibodies that are otherwise unable to mediate these activities. [ABSTRACT FROM AUTHOR]

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibodydependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy. [ABSTRACT FROM AUTHOR]

The envelope glycoproteins (Envs) from human immunodeficiency virus type 1 (HIV-1) mediate viral entry. The binding of the HIV-1 gp120 glycoprotein to CD4 triggers conformational changes in gp120 that allow high-affinity binding to its coreceptors. In contrast to all other Envs from the same phylogenetic group, M, which possess a serine (S) at position 375, those from CRF01_AE strains possess a histidine (H) at this location. This residue is part of the Phe43 cavity, where residue 43 of CD4 (a phenylalanine) engages with gp120. Here we evaluated the functional consequences of replacing this residue in two CRF01_AE Envs (CM244 and 92TH023) by a serine. We observed that reversion of amino acid 375 to a serine (H375S) resulted in a loss of functionality of both CRF01_AE Envs as measured by a dramatic loss in infectivity and ability to mediate cell-to-cell fusion. While no effects on processing or trimer stability of these variants were observed, decreased functionality could be linked to a major defect in CD4 binding induced by the replacement of H375 by a serine. Importantly, mutations of residues 61 (layer 1), 105 and 108 (layer 2), and 474 to 476 (layer 3) of the CRF01_AE gp120 inner domain layers to the consensus residues present in group M restored CD4 binding and wild-type levels of infectivity and cell-to-cell fusion. These results suggest a functional coevolution between the Phe43 cavity and the gp120 inner domain layers. Altogether, our observations describe the functional importance of amino acid 375H in CRF01_AE envelopes. [ABSTRACT FROM AUTHOR]

SERINC5 is able to restrict HIV-1 infection by drastically impairing the infectivity of viral particles. Studies have shown that the HIV-1 Nef protein counters SERINC5 through downregulating SERINC5 from the cell surface and preventing the virion incorporation of SERINC5. In addition, the Env proteins of some HIV-1 strains can also overcome SERINC5 inhibition. However, it is unclear how HIV-1 Env does so and why HIV-1 has two mechanisms to resist SERINC5 inhibition. The results of this study show that neither Env nor Nef prevents high levels of ectopic SERINC5 from being incorporated into HIV-1 particles, except that Env, but not Nef, is able to resist inhibition by virion-associated SERINC5. Testing of a panel of HIV-1 Env proteins from different subtypes revealed a high frequency of SERINC5-resistant Envs. Interestingly, although the SERINC5-bearing viruses were not inhibited by SERINC5 itself, they became more sensitive to the CCR5 inhibitor maraviroc and some neutralizing antibodies than the SERINC5-free viruses, which suggests a possible influence of SERINC5 on Env function. We conclude that HIV-1 Env is able to overcome SERINC5 without preventing SERINC5 virion incorporation. [ABSTRACT FROM AUTHOR]

Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20 Å, native MLV Env presents as a hollow trimer (height ∼85 Å, diameter ∼120 Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35 Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition. [ABSTRACT FROM AUTHOR]

In 2009, Dimitrov's group reported that the inferred germline ( iGL) forms of several HIV-1 broadly neutralizing antibodies ( bNAbs) did not display measurable binding to a recombinant gp140 Env protein (derived from the dual-tropic 89.6 virus), which was efficiently recognized by the mature (somatically mutated) antibodies. At that time, a small number of bNAbs were available, but in the following years, the implementation of high-throughput B-cell isolation and sequencing assays and of screening methodologies facilitated the isolation of greater numbers of bNAbs from infected subjects. Using these newest bNAbs, and a wide range of diverse recombinant Envs, we and others confirmed the observations made by Dimitrov's group. The results from these studies created a paradigm shift in our collective thinking as to why recombinant Envs are ineffective in eliciting bNAbs and has led to the 'germline-targeting' immunization approach. Here we discuss this approach in detail: what has been done so far, the advantages and limitations of the current germline-targeting immunogens and of the animal models used to test them, and we conclude with a few thoughts about future directions in this area of research. [ABSTRACT FROM AUTHOR]

Guanylate binding proteins (GBPs) are interferon-inducible cellular factors that belong to the superfamily of guanosine triphosphatases (GTPases) and play important roles in the cell-intrinsic defense against bacteria, protozoa and viruses. In a recent report in Cell Host & Microbe, we identify GBP5 as novel restriction factor of HIV-1 that reduces the infectivity of progeny virions by interfering with processing and incorporation of the viral envelope (Env) glycoprotein. The inhibitory activity of GBP5 requires C-terminal isoprenylation, mediating Golgi-association, but not its GTPase function. Notably, GBP5 expression levels vary considerably in human macrophages and inversely correlate with infectious virus yield. We demonstrate that GBP5 can be evaded by an unusual tradeoff mechanism: Naturally occurring mutations in the start codon of the viral accessory genevpuattenuate GBP5 inhibition by increasing Env expression at the cost of Vpu function. Whether direct counteraction mechanisms or more subtle changes balancing Vpu and Env expression also affect HIV-1 inhibition by GBP5 remains to be clarified. Other open questions are whether GBP5 restricts HIV-1 in CD4+T cells and if other GBP family members also decrease infectivity of HIV and/or additional enveloped viruses. [ABSTRACT FROM AUTHOR]

The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies., Competing Interests: The authors declare no conflict of interest.

Background: Entry of human immunodeficiency virus type 1 (HIV-1) into cells involves the interaction of the viral gp120 envelope glycoproteins (Env) with cellular CD4 and a secondary coreceptor, which is typically one of the chemokine receptors CCR5 or CXCR4. CCR5-using (R5) HIV-1 strains that display reduced sensitivity to CCR5 antagonists can use antagonist-bound CCR5 for entry. In this study, we investigated whether naturally occurring gp120 alterations in HIV-1 subtype C (C-HIV) variants exist in antiretroviral therapy (ART)-naïve subjects that may influence their sensitivity to the CCR5 antagonist maraviroc (MVC). Results: Using a longitudinal panel of 244 R5 Envs cloned from 20 ART-naïve subjects with progressive C-HIV infection, we show that 40% of subjects (n = 8) harbored viruses that displayed incomplete inhibition by MVC, as shown by plateau's of reduced maximal percent inhibitions (MPIs). Specifically, when pseudotyped onto luciferase reporter viruses, 16 Envs exhibited MPIs below 98% in NP2-CCR5 cells (range 79.7-97.3%), which were lower still in 293-Affinofile cells that were engineered to express high levels of CCR5 (range 15.8-72.5%). We further show that Envs exhibiting reduced MPIs to MVC utilized MVC-bound CCR5 less efficiently than MVC-free CCR5, which is consistent with the mechanism of resistance to CCR5 antagonists that can occur in patients failing therapy. Mutagenesis studies identified strain-specific mutations in the gp120 V3 loop that contributed to reduced MPIs to MVC. Conclusions: The results of our study suggest that some ART-naïve subjects with C-HIV infection harbor HIV-1 with reduced MPIs to MVC, and demonstrate that the gp120 V3 loop region contributes to this phenotype. [ABSTRACT FROM AUTHOR]

Background and Objective: Elephantiasis nostras verrucosa (ENV) is a disfiguring skin condition that is difficult to treat. Existing treatment modalities serve to improve cosmesis or treat symptoms. Herein, we report a case of ENV with lymphocutaneous fistula successfully treated with ablative carbon dioxide laser. Study Design/Patients and Methods: A 57-year-old woman with biopsy-proven ENV with lymphocutaneous fistula was treated with ablative carbon dioxide laser to the symptomatic area of her right thigh in 3 treatment sessions over 6 months. Results: The patient had resolution of lymphocutaneous drainage as well as 90% improvement in the appearance of ENV lesions at the 1-month follow-up visit. Conclusion: Ablative carbon dioxide laser may provide cosmetic, symptomatic, and medical benefit for patients with localized ENV. [ABSTRACT FROM AUTHOR]

Apoptosis of uninfected bystander cells is a key element of HIV pathogenesis and believed to be the driving force behind the selective depletion of CD4+ T cells leading to immunodeficiency. While several viral proteins have been implicated in this process the complex interaction between Env glycoprotein expressed on the surface of infected cells and the receptor and co-receptor expressing bystander cells has been proposed as a major mechanism. HIV-1 utilizes CD4 as the primary receptor for entry into cells; however, it is the viral co-receptor usage that greatly influences CD4 decline and progression to AIDS. This phenomenon is relatively simple for X4 viruses, which arise later during the course of the disease, are considered to be highly fusogenic, and cause a rapid CD4+ T cell decline. However, in contrast, R5 viruses in general have a greater transmissibility, are encountered early during the disease and have a lesser pathogenic potential than the former. The above generalization gets complicated in numerous situations where R5 viruses persist throughout the disease and are capable of causing a rigorous CD4+ T cell decline. This review will discuss the multiple factors that are reported to influence HIV induced bystander apoptosis and pathogenesis including Env glycoprotein phenotype, virus tropism, disease stage, co-receptor expression on CD4+ T cells, immune activation and therapies targeting the viral envelope.

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV‑induced pathogenesis, Env transformation, and other aspects of JSRV biology.

Indeterminate results of serological HIV test have been found in Indonesia. The screening procedure is following the prescribed by WHO for screening of blood donors which is based on 3 different serological HIV test during screening of blood donors. Discordant results are interpreted as indeterminate. In order to find the cause of the indeterminate results, we conducted RT-PCR detection of HIV RNA in samples from blood donors with indeterminate serological HIV-test. Plasma samples showing indeterminate results by serological testing from blood donor were collected from Blood Transfusion Service of Indonesian Red Cross and subjected to confirmatory test by using western blot. Test of 40 samples showed that 90% of the samples remain indeterminate. Most of the indeterminate results were caused by the reactivity towards p24 antigen. RT-PCR targeted at LTR, pol, env and p24 region of HIV was performed to detect the presence of HIV RNA. Primer were consequently designed to amplify these regions. Preliminary results showed 1/35 24/32 (75%) positive LTR, 4/31 (12%) positive pol and 1/3 (33%) positive env. Amplification in p24 region showed that bands found have lower size than expected. Sequencing was performed to confirm these findings. The results suggest that further exploration should be performed in order to understand the magnitude of problem regarding HIV-RNA positivity in indeterminate sample by serological test to determine future direction such as the necessity to obtain information regarding the influence of circulating HIV strain in Indonesian population towards accuracy of HIV diagnostics.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of “classical” vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Up to 25 percent of all anthropogenic greenhouse gas emissions are caused by deforestation, and Indonesia is the third largest greenhouse gas emitter worldwide due to land use change and deforestation. On the island of Sulawesi in the vicinity of the Lore Lindu National Park (LLNP), many smallholders contribute to conversion processes at the forest margin as a result of their agricultural practices. Specifically the area dedicated to cocoa plantations has increased from zero (1979) to nearly 18,000 hectares (2001). Some of these plots have been established inside the 220,000 hectares of the LLNP. An intensification process is observed with a consequent reduction of the shade tree density. This study assesses which impact carbon sequestration payments for forest management systems have on the prevailing land use systems. Additionally, the level of incentives is determined which motivates farmers to desist from further deforestation and land use intensification activities. Household behaviour and resource allocation is analysed with a comparative static linear programming model. As these models prove to be a reliable tool for policy analysis, the output can indicate the adjustments in resource allocation and land use shifts when introducing compensation payments. The data was collected in a household survey in six villages around the LLNP. Four household categories are identified according to their dominant agroforestry systems. These range from low intensity management with a high degree of shading to highly intensified systems with no shade cover. At the plot level, the payments required for inducing the adoption of more sustainable land use practices are the highest for the full shade cocoa agroforestry system, but with low carbon prices of €5 tCO2e-1 these constitute 5 percent of the cocoa gross margin. Focusing on the household level, however, an increase up to 18 percent of the total gross margin can be realised. Furthermore, for differentiated carbon prices up to €32 tCO2e-1 the majority of the households have an incentive to adopt the more sustainable shade intensive agroforestry system. Additionally, the results show that the deforestation activities of most households could be stopped with current carbon prices.

The commons literature makes much of the changes within the traditional land use sectors of developed countries. This largely focuses on the decline of the economic function of commons that threaten their existence, the emergence of multiple use patterns, and the resilience and policy adaptation needed to continue. The situation in England and Wales is used to illustrate that commons are increasingly important to a number of ‘new’ rural functions and that the associated policy developments may hold an important message for progress towards sustainable multifunctional land management more generally. This article reviews and updates what is meant by the term common land within England and Wales, while outlining its current importance and threats. The commons literature is investigated to see if the approach is useful in revealing the current issues associated with the incorporation of new stakeholders and functions within a traditional structure. Recent changes and developments surrounding the Commons Act 2006 are assessed to see if they are likely to assist in sustaining these commons through the twenty-first century. The article argues that any new approach requires long term planning and a commitment to support local participation among commoners and others who are involved in the governance and management of these areas of land. In order for these challenges to be met there needs to be an understanding of the functions and cultural traditions of common land as well as of the changes in society associated with the decline in traditional agrarian management in developed countries. Such challenges can rarely if ever be achieved through legislation and policy developments, requiring an investment in developing locally based solutions.