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Mojiang virus (MojV) is the first henipavirus identified in a rodent and known only by sequence data, whereas all other henipaviruses have been isolated from bats (Hendra virus, Nipah virus, Cedar virus) or discovered by sequence data from material of bat origin (Ghana virus). Ephrin-B2 and -B3 are entry receptors for Hendra and Nipah viruses, but Cedar virus can utilize human ephrin-B1, -B2, -A2 and -A5 and mouse ephrin-A1. However, the entry receptor for MojV remains unknown, and its species tropism is not well characterized. Here, we utilized recombinant full-length and soluble forms of the MojV fusion (F) and attachment (G) glycoproteins in membrane fusion and receptor tropism studies. MojV F and G were functionally competent and mediated cell-cell fusion in primate and rattine cells, albeit with low levels and slow fusion kinetics. Although a relative instability of the pre-fusion conformation of a soluble form of MojV F was observed, MojV F displayed significantly greater fusion activity when heterotypically paired with Ghana virus G. An exhaustive investigation of A- and B-class ephrins indicated that none serve as a primary receptor for MojV. The MojV cell fusion phenotype is therefore likely the result of receptor restriction rather than functional defects in recombinant MojV F and G glycoproteins.

Several mammarenaviruses can cause deadly hemorrhagic fever infections in humans, with limited preventative and therapeutic measures available. Arenavirus cell entry is mediated by the viral glycoprotein (GP) complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The GP2 cytoplasmic tail (CT) is relatively conserved among arenaviruses and is known to interact with the SSP to regulate GP processing and membrane fusion, but its biological role in the context of an infectious virus has not been fully characterized. Using a Pichinde virus (PICV) GP expression vector and a PICV reverse genetics system, we systematically characterized the functional roles of 12 conserved residues within the GP2 CT in GP processing, trafficking, assembly, and fusion, as well as in viral replication. Except for P478A and K505A R508A, alanine substitutions at conserved residues abolished GP processing and membrane fusion in plasmid-transfected cells. Six invariant H and C residues and W503 are essential for viral replication, as evidenced by the fact that their mutant viruses could not be rescued. Both P480A and R482A mutant viruses were rescued, grew similarly to wild-type (WT) virus, and produced evidently processed GP1 and GP2 subunits in virus-infected cells, despite the fact that the same mutations abolished GP processing and membrane fusion in a plasmid-based protein expression system, illustrating the importance of using an infectious-virus system for analyzing viral glycoprotein function. In summary, our results demonstrate an essential biological role of the GP2 CT in arenavirus replication and suggest it as a potential novel target for developing antivirals and/or attenuated viral vaccine candidates. IMPORTANCE Several arenaviruses, such as Lassa virus (LASV), can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapeutics are available. Viral entry is mediated by the arenavirus GP complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The cytoplasmic tail (CT) of GP2 is highly conserved among arenaviruses, but its functional role in viral replication is not completely understood. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we show that the GP2 CT contains certain conserved residues that are essential for virus replication, implicating it as a potentially good target for developing antivirals and live-attenuated viral vaccines against deadly arenavirus pathogens., (Copyright © 2019 American Society for Microbiology.)

Oncolytic herpes simplex viruses (oHSVs) have been approved for clinical usage and become more and more popular for tumor virotherapy. However, there are still many issues for the oHSVs used in clinics and clinical trials. The main issues are the limited anti-tumor effects, intratumor injection, and some side effects. To overcome such challenges, here we review the genetic engineering of the envelope glycoproteins for oHSVs to target tumors specifically, and at the same time we summarize the many neutralization antibodies against the envelope glycoproteins and align the neutralization epitopes with functional domains of the respective glycoproteins for future identification of new functions of the glycoproteins and future engineering of the epitopes to escape from host neutralization.

Female genital epithelial cells cover the genital tract and provide the first line of protection against infection with sexually transmitted pathogenic viruses. These cells normally are impervious to HIV-1. We report that coinfection of cells by HIV-1 and another sexually transmitted virus, human T-lymphotropic virus 1 (HTLV-1), led to production of HIV-1 that had expanded cell tropism and was able to directly infect primary vaginal and cervical epithelial cells. HIV-1 infection of epithelial cells was blocked by neutralizing antibodies against the HTLV-1 envelope (Env) protein, indicating that the infection was mediated through HTLV-1 Env pseudotyping of HIV-1. Active replication of HIV-1 in epithelial cells was demonstrated by inhibition with anti-HIV-1 drugs. We demonstrated that HIV-1 derived from peripheral blood of HIV-1-HTLV-1-coinfected subjects could infect primary epithelial cells in an HTLV-1 Env-dependent manner. HIV-1 from subjects infected with HIV-1 alone was not able to infect epithelial cells. These results indicate that pseudotyping of HIV-1 with HTLV-1 Env can occur in vivo Our data further reveal that active replication of both HTLV-1 and HIV-1 is required for production of pseudotyped HIV-1. Our findings indicate that pseudotyping of HIV-1 with HTLV-1 Env in coinfected cells enabled HIV-1 to directly infect nonpermissive female genital epithelial cells. This phenomenon may represent a risk factor for enhanced sexual transmission of HIV-1 in regions where virus coinfection is common. IMPORTANCE Young women in certain regions of the world are at very high risk of acquiring HIV-1, and there is an urgent need to identify the factors that promote HIV-1 transmission. HIV-1 infection is frequently accompanied by infection with other pathogenic viruses. We demonstrate that coinfection of cells by HIV-1 and HTLV-1 can lead to production of HIV-1 pseudotyped with HTLV-1 Env that is able to directly infect female genital epithelial cells both in vitro and ex vivo Given the function of these epithelial cells as genital mucosal barriers to pathogenic virus transmission, the ability of HIV-1 pseudotyped with HTLV-1 Env to directly infect female genital epithelial cells represents a possible factor for increased risk of sexual transmission of HIV-1. This mechanism could be especially impactful in settings such as Sub-Saharan Africa and South America, where HIV-1 and HTLV-1 are both highly prevalent., (Copyright © 2018 Tang et al.)

Background & Objectives: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV., Methods: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015., Results: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes., Interpretation & Conclusions: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now., Competing Interests: None

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. IMPORTANCE The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here, we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers., (Copyright © 2017 American Society for Microbiology.)

Background: Analysis of codon usage bias is an extremely versatile method using in furthering understanding of the genetic and evolutionary paths of species. Codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) has remained largely unexplored at present. Hence, the codon usage bias of NPV envelope glycoprotein was analyzed here to reveal the genetic and evolutionary relationships between different viral species in baculovirus genus., Results: A total of 9236 codons from 18 different species of NPV of the baculovirus genera were used to perform this analysis. Glycoprotein of NPV exhibits weaker codon usage bias. Neutrality plot analysis and correlation analysis of effective number of codons (ENC) values indicate that natural selection is the main factor influencing codon usage bias, and that the impact of mutation pressure is relatively smaller. Another cluster analysis shows that the kinship or evolutionary relationships of these viral species can be divided into two broad categories despite all of these 18 species are from the same baculovirus genus., Conclusions: There are many elements that can affect codon bias, such as the composition of amino acids, mutation pressure, natural selection, gene expression level, and etc. In the meantime, cluster analysis also illustrates that codon usage bias of virus envelope glycoprotein can serve as an effective means of evolutionary classification in baculovirus genus.

Systemic autoimmune diseases (SADs) are a growing spectrum of autoimmune disorders that commonly affect multiple organs. The role of Epstein–Barr virus (EBV) infection or reactivation as a trigger for the initiation and progression of SADs has been established, while the relationship between EBV envelope glycoproteins and SADs remains unclear. Here, we assessed the levels of IgG, IgA, and IgM against EBV glycoproteins (including gp350, gp42, gHgL, and gB) in serum samples obtained from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and found that RA and SLE patients exhibited a statistically significant increase in the levels of 8 and 11 glycoprotein antibodies, respectively, compared to healthy controls (p < 0.05). The LASSO model identified four factors as significant diagnostic markers for RA: gp350 IgG, gp350 IgA, gHgL IgM, and gp42 IgA; whereas for SLE it included gp350 IgG, gp350 IgA, gHgL IgA, and gp42 IgM. Combining these selected biomarkers yielded an area under the curve (AUC) of 0.749 for RA and 0.843 for SLE. We subsequently quantified the levels of autoantibodies associated with SADs in mouse sera following immunization with gp350. Remarkably, none of the tested autoantibody levels exhibited statistically significant alterations. Elevation of glycoprotein antibody concentration suggests that Epstein–Barr virus reactivation and replication occurred in SADs patients, potentially serving as a promising biomarker for diagnosing SADs. Moreover, the absence of cross‐reactivity between gp350 antibodies and SADs‐associated autoantigens indicates the safety profile of a vaccine based on gp350 antigen. [ABSTRACT FROM AUTHOR]

human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (aids), a pandemic with high economic and social costs. The envelope glycoprotein (env) of the virus mediates the infectious process by binding to and entering the host cell, one of the main target components of studies since its discovery. Its endodomain or C-terminal tail (CTT) participates in late replicative cycle processes, such as intracellular trafficking, activation, and cell death, which occurs because it interacts with multiple cellular factors through motifs or signal sequences present throughout its structure. Although these interactions have not been fully understood at specific levels, studies over more than three decades leave no doubt that this domain plays a fundamental role in the biology of the virus and probably the development of the disease. This review describes the studies carried out to date that demonstrate the importance of the CTT, focusing on the motifs responsible for its interactions and its possible roles in the pathogenicity of the infection. [ABSTRACT FROM AUTHOR]

Highlights • The deletion of v-cath/chiA gene enhances HIV gp140 expression yield in insect cell. • gp140 mimicked the native-like trimer and adopted distinctive glycosylation pattern. • The native-like Env proteins elicited cross-clade neutralizing antibody in mice. • The alternative gp140 production way might benefit for HIV vaccine development. Abstract The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13–15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Three peptides derived from envelope glycoprotein were evaluated for inhibiting the viral infection. • Pep2 showed highest percentage of inhibitory activity towards, DENV-1, DENV-2, DENV-3 and DENV-4. • The synthetic peptides inhibited viral entry by binding to the envelope glycoprotein of Dengue Virus. Abstract Dengue fever is the most prevalent arthropod-borne viral disease, and no specific therapeutic or promising antiviral drug is available for its treatment. Peptide inhibitors are less toxic than synthetic compounds and have found proven effective against viral infections. Here, three peptides that mimic part of the E protein of the dengue virus (DENV) were synthesized and evaluated for their inhibitory activity against four serotypes of DENV in African green monkey kidney (Vero) and rhesus macaque (Macaca mulatta) monkey kidney (LLC-MK2) cell lines. The three peptides, Pep1, Pep2, and Pep3 are located in domains I, II, and III of the E protein respectively. All three peptides effectively reduced >80% of focus forming units in the virus treated mammalian cell lines than control and exhibited their IC 50 in the range of 10–33 μM. Pep1 was found effective against DENV-2, DENV-3, and DENV-4 (IC 80 below 50 μM). Pep2 showed the highest inhibitory activity against all four serotypes (IC 50 below 20 μM). Pep3 reduced the 80% focus forming units in all serotypes at the concentration of 40 μM. Evaluation of peptides at different time points of viral infection in the mammalian cell lines revealed that the peptides inhibited viral infection by binding to the virus and not by binding to cellular receptors and blocking viral entry. The peptides assumedly exert their inhibitory effects by binding to the E protein and repressing its conformational changes; this prevents the virus from binding to cellular receptors, thereby inhibiting viral entry. Hence, these peptides might limit viral spread and reduce the virus's ability to infect. [ABSTRACT FROM AUTHOR]

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve the opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad repeat regions (HR1 and HR2) first into an extended prehairpin intermediate and then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that contribute not only to fusion inhibitor resistance and 6HB stability but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs), we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env. [ABSTRACT FROM AUTHOR]

Background: Small-molecule CD4-mimetic compounds (CD4mc) inhibit human immunodeficiency virus (HIV-1) entry by blocking binding to the CD4 receptor and by premature triggering of the viral envelope glycoprotein (Env) spike.Methods: The efficacy of a CD4mc in protecting bone marrow-liver-thymus (BLT) humanized mice from vaginal HIV-1 challenge was evaluated.Results: Intravaginal application of the CD4mc JP-III-48, either before or simultaneously with virus challenge, protected BLT humanized mice from HIV-1JR-CSF infection in a dose- dependent manner.Conclusion: The direct antiviral effects of a CD4mc prevent HIV-1 infection in a murine model of sexual transmission. [ABSTRACT FROM AUTHOR]

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ~10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies. IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env. [ABSTRACT FROM AUTHOR]

HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities. IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein. [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 envelope glycoprotein of diverse endogenous and exogenous retroviruses is considered inherently immunosuppressive. Extensive work mapped the immunosuppressive activity to a highly conserved domain, termed the immunosuppressive domain (ISD), in the transmembrane (TM) subunit of the envelope glycoprotein and identified two naturally polymorphic key residues that afford immunosuppressive activity to distinct envelope glycoproteins. Concurrent mutation of these two key residues (E14R and A20F) in the envelope glycoprotein of the Friend murine leukemia virus (F-MLV) ISD has been reported to abolish its immunosuppressive activity, without affecting its fusogenicity, and to weaken the ability of the virus to replicate specifically in immunocompetent hosts. Here, we show that mutation of these key residues did, in fact, result in a substantial loss of F-MLV infectivity, independently of host immunity, challenging whether associations exist between the two. Notably, a loss of infectivity incurred by the F-MLV mutant with the E14R and A20F double ISD mutation was conditional on expression of the ecotropic envelope receptor murine cationic amino acid transporter-1 (mCAT1) in the virus-producing cell. Indeed, the F-MLV mutant retained infectivity when it was produced by human cells, which naturally lack mCAT1 expression, but not by murine cells. Furthermore, mCAT1 overexpression in human cells impaired the infectivity of both the F-MLV double mutant and the wild-type F-MLV strain, suggesting a finely tuned relationship between the levels of mCAT1 in the producer cell and the infectivity of the virions produced. An adverse effect on this relationship, rather than disruption of the putative ISD, is therefore more likely to explain the loss of F-MLV infectivity incurred by mutations in key ISD residues E14 and A20. [ABSTRACT FROM AUTHOR]

The extent of virus transmission among individuals and species is generally determined by the presence of specific membraneembedded virus receptors required for virus entry. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor is the first and crucial step in determining host specificity. Using a well-established retroviral model--avian Rous sarcoma virus (RSV)--we analyzed changes in an RSV variant that had repeatedly been able to infect rodents. By envelope gene (env) sequencing, we identified eight mutations that do not match the already described mutations influencing the host range. Two of these mutations--one at the beginning (D32G) of the surface Env subunit (SU) and the other at the end of the fusion peptide region (L378S)--were found to be of critical importance, ensuring transmission to rodent, human, and chicken cells lacking the appropriate receptor. Furthermore, we carried out assays to examine the virus entry mechanism and concluded that these two mutations cause conformational changes in the Env variant and that these changes lead to an activated, or primed, state of Env (normally induced after Env interaction with the receptor). In summary, our results indicate that retroviral host range extension is caused by spontaneous Env activation, which circumvents the need for original cell receptor. This activation is, in turn, caused by mutations in various env regions. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility. [ABSTRACT FROM AUTHOR]

BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail. [ABSTRACT FROM AUTHOR]

Background: Current HIV-1 immunogens are unable to induce antibodies that can neutralize a broad range of HIV-1 (broadly neutralizing antibodies; bNAbs). However, such antibodies are elicited in 10-30% of HIV-1 infected individuals, and the co-evolution of the virus and the humoral immune responses in these individuals has attracted attention, because they can provide clues for vaccine design. Results: Here we characterized the NAb responses and envelope glycoprotein evolution in an HIV-1 infected "elite neutralizer" of the Amsterdam Cohort Studies on HIV-1 infection and AIDS who developed an unusually potent bNAb response rapidly after infection. The NAb response was dependent on the N332-glycan and viral resistance against the N332-glycan dependent bNAb PGT135 developed over time but viral escape did not occur at or near this glycan. In contrast, the virus likely escaped by increasing V1 length, with up to 21 amino acids, accompanied by the introduction of 1-3 additional glycans, as well as 2-4 additional cysteine residues within V1. Conclusions: In the individual studied here, HIV-1 escaped from N332-glycan directed NAb responses without changing the epitope itself, but by elongating a variable loop that shields this epitope. [ABSTRACT FROM AUTHOR]

Background: Four antigenically distinct serotypes (1-4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype. Results: We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies. Conclusions: This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes. [ABSTRACT FROM AUTHOR]

Background: Broadly neutralizing antibodies (bnAbs) directed against the mannose-patch on the HIV envelope glycoprotein gp120 have several features that make them desirable targets for vaccine design. The PGT125-131 bnAb family is of particular interest due to its superior breadth and potency. The overlapping epitopes recognized by this family are intricate and neutralization requires interaction with at least two N-linked glycans (N332/N334, N295 or N301) in addition to backbone-mediated contact with the 323IGDIR327 motif of the V3 loop. We have recently shown that this bnAb family consists of two distinct antibody classes that can bind alternate arrangements of glycans in the mannose-patch in the absence of N332 thereby limiting viral escape. This led us to further investigate viral resistance and escape mechanisms to the PGT125-131 bnAb family. Results: Using an escape virus isolated from the PGT125-131 donor as a guide, we show that mutating both the V3 core protein epitope and repositioning critical N-linked glycosylation sites are required to restore neutralization sensitivity. Interestingly, neutralization sensitivity could be restored via different routes for the two distinct bnAb classes within the PGT125-131 family, which may have been important in generating the divergence in recognition. We demonstrate that the observed V3 mutations confer neutralization resistance in other virus strains through both gain-of-function and escape studies. Furthermore, we show that the V3 loop is important in facilitating promiscuous binding to glycans within the mannose-patch. Conclusions: These data highlight the importance of the V3 loop in the design of immunogens aimed at inducing broad and potent bnAbs that can bind promiscuously to the mannose-patch. [ABSTRACT FROM AUTHOR]

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations of mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus "hot-spots" under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. With well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent "cocktail" vaccines. [ABSTRACT FROM AUTHOR]

Background: Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation. Findings: We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-bearing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses (but not significantly), to several tier 1B viruses and the autologous tier 2 virus than when the same trimers were delivered as soluble proteins. Conclusions: This or other nanoparticle designs may be practical ways to improve the immunogenicity of envelope glycoprotein trimers. [ABSTRACT FROM AUTHOR]

Background: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env. Results: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps. Conclusions: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions. [ABSTRACT FROM AUTHOR]

Three bovine monoclonal antibodies (BomAb) raised to bovine herpesvirus (BoHV) 1.1 and specific for the viral glycoproteins gB, gC, and gD were tested for reactivity to two isolates of BoHV-1.1, one of BoHV-1.2, and two of BoHV-5 in virus neutralization and indirect fluorescent antibody assays. They were also tested with other herpesviruses infecting cattle and other mammalian alphaherpesviruses, and found negative or of negligible reactivity. Their BoHV-1.1 epitope specificity was examined using competitive ELISA with peroxidase-labeled murine monoclonal antibodies (MumAb) that had been previously characterized. To explain the incongruities observed, the amino acid sequences of the epitopes and adjacent regions of BoHV-1.1, 1.2, and 5 were compared, and molecular modeling was performed using human herpesvirus 1 glycoprotein crystals as templates. The anti-gB BomAb reacted strongly with BoHV-1.1 and BoHV-1.2, and poorly or not at all with BoHV-5. It competed with a MumAb specific for a BoHV-1.1 gB epitope previously shown to only partially cross-react between BoHV-1 and BoHV-5. BoHV-5 gB has nearly identical sequence with BoHV-1.1 in the epitope region, but modeling suggested the lack of cross-reactivity of the MumAb was due to masking of the epitope in BoHV-5 by an adjacent region, which has significant sequence differences between BoHV-1.1 and BoHV-5. The BomAb reactivity could also be explained by masking, or by reactivity with the adjacent region. The anti-gC BomAb reacted strongly with one isolate of BoHV-1.1 and BoHV-1.2, less well with a heterologous isolate of BoHV-1.1, and poorly or not at all with BoHV-5. It did not compete with any of the anti-gC MumAb tested, but a target domain was suggested by BoHV-1.1, 1.2, and 5 sequence divergence. The anti-gD BomAb reacted strongly with all BoHV-1.1, 1.2, and 5 isolates tested. However, it competed with two MumAb previously shown to not cross-react between BoHV-1.1 and BoHV-5. Sequence analysis and modeling suggested the cross-reactivity of the anti-gD BomAb was due to it reacting with an epitope-adjacent region or regions conserved between BoHV-1.1 and BoHV-5, but not with other alphaherpesviruses. The results suggest the usefulness of combining in vitro biological data with sequence or structure modeling data to investigate important epitopes involved in immunity to infectious agents. [ABSTRACT FROM AUTHOR]

Background The CD4 binding site (CD4bs) of envelope glycoprotein (Env) gp120 is a functionally conserved, important target of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Two neutralizing human monoclonal antibodies, IgG1 b12 (b12) and VRC01, are broadly reactive neutralizing antibodies which recognize conformational epitopes that overlap the CD4bs of Env gp120; however, many CRF01_AE viruses are resistant to neutralization mediated by these antibodies. We examined the mechanism underlying the b12 resistance of the viruses using CRF01_AE Env (AE-Env)-recombinant viruses in this study. Results Our results showed that an amino acid substitution at position 185 in the V2 region of gp120 played a crucial role in regulating the b12 susceptibility of AE-Env-recombinant viruses by cooperating with 2 previously reported potential N-linked glycosylation (PNLG) sites at positions 186 (N186) and 197 (N197) in the V2 and C2 regions of Env gp120. The amino acid residue at position 185 and 2 PNLG sites were responsible for the b12 resistance of 21 of 23 (>91%) AE-Env clones tested. Namely, the introduction of aspartic acid at position 185 (D185) conferred b12 susceptibility of 12 resistant AE-Env clones in the absence of N186 and/or N197, while the introduction of glycine at position 185 (G185) reduced the b12 susceptibility of 9 susceptible AE-Env clones in the absence of N186 or N197. In addition, these amino acid mutations altered the VRC01 susceptibility of many AE-Env clones. Conclusions We propose that the V2 and C2 regions of AE-Env gp120 contain the major determinants of viral resistance to CD4bs antibodies. CRF01_AE is a major circulating recombinant form of HIV-1 prevalent in Southeast Asia. Our data may provide important information to understand the molecular mechanism regulating the neutralization susceptibility of CRF01_AE viruses to CD4bs antibodies. [ABSTRACT FROM AUTHOR]

Highlights: [•] Viral membrane fusion proteins fall into three distinct structural classes (I–III). [•] In 2013, the E2 glycoproteins of pesti- and hepaciviruses were found to have novel folds. [•] E1 and E2 proteins of pesti- and hepaciviruses define a new class of fusion machinery. [•] Structural data suggest that fusion proteins evolved by host–virus or virus–virus transfer. [ABSTRACT FROM AUTHOR]

Background: Current HIV-1 envelope glycoprotein (Env) vaccines are unable to induce cross-reactive neutralizing antibodies. However, such antibodies are elicited in 10-30% of HIV-1 infected individuals, but it is unknown why these antibodies are induced in some individuals and not in others. We hypothesized that the Envs of early HIV-1 variants in individuals who develop cross-reactive neutralizing activity (CrNA) might have unique characteristics that support the induction of CrNA. Results: We retrospectively generated and analyzed env sequences of early HIV-1 clonal variants from 31 individuals with diverse levels of CrNA 2-4 years post-seroconversion. These sequences revealed a number of Env signatures that coincided with CrNA development. These included a statistically shorter variable region 1 and a lower probability of glycosylation as implied by a high ratio of NXS versus NXT glycosylation motifs. Furthermore, lower probability of glycosylation at position 332, which is involved in the epitopes of many broadly reactive neutralizing antibodies, was associated with the induction of CrNA. Finally, Sequence Harmony identified a number of amino acid changes associated with the development of CrNA. These residues mapped to various Env subdomains, but in particular to the first and fourth variable region as well as the underlying α2 helix of the third constant region. Conclusions: These findings imply that the development of CrNA might depend on specific characteristics of early Env. Env signatures that correlate with the induction of CrNA might be relevant for the design of effective HIV-1 vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s). [Copyright &y& Elsevier]

Abstract: The need for an effective vaccine to prevent the global spread of human immunodeficiency virus type 1 (HIV-1) is well recognized. Passive immunization and challenge studies in non-human primates testify that broadly neutralizing antibodies (BrNAbs) can accomplish protection against infection. In recent years, the introduction of new techniques has facilitated the discovery of an unprecedented number of new human BrNAbs that target and delineate diverse conserved epitopes on the envelope glycoprotein spike (Env). The epitopes of these BrNAbs can serve as templates for immunogen design aimed to induce similar antibodies. Here we will review the characteristics of the different classes of BrNAbs and their target epitopes, as well as factors associated with their development and implications for vaccine design. [Copyright &y& Elsevier]

Abstract: GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV. [Copyright &y& Elsevier]

Abstract: The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated. [Copyright &y& Elsevier]

Abstract: The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46mg per larva, respectively, at 6days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen. [Copyright &y& Elsevier]

Abstract: We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens. [Copyright &y& Elsevier]

Background: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20-57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region. [ABSTRACT FROM AUTHOR]

Arenaviruses comprise a diverse family of enveloped negative-strand RNA viruses that are endemic to specific rodent hosts worldwide. Several arenaviruses cause severe hemorrhagic fevers in humans, including Junín and Machupo viruses in South America and Lassa fever virus in western Africa. Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins. We will review the curious properties of the tripartite GPC complex and describe evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention. [ABSTRACT FROM AUTHOR]

Abstract: We recently described a Venezuelan equine encephalitis virus (VEEV)-specific human monoclonal antibody (MAb), F5 nIgG, that recognizes a new neutralization epitope on the VEEV E2 envelope glycoprotein. In this study, we investigated the ability of F5 nIgG given prophylactically or therapeutically to protect mice from subcutaneous or aerosolized VEEV infection. F5 nIgG had potent ability to protect mice from infection by either route when administered 24h before exposure; however, mice treated 24h after aerosol exposure developed central nervous system infections but exhibited no clinical signs of disease. Infectious virus, viral antigen and RNA were detected in brains of both treated and untreated mice 2–6days after aerosol exposure but were cleared from the brains of treated animals by 14–28days after infection. This fully human MAb could be useful for prophylaxis or immediate therapy for individuals exposed to VEEV accidentally in the laboratory or during a deliberate release. [Copyright &y& Elsevier]

Abstract: Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP1,2 or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP1,2 peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Taï Forest viruses and a mucin-like domain-deleted Sudan virus GP1,2 (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP1,2 and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP1,2, but not particles pseudotyped with other ebolavirus GP1,2. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP1,2, as well as analysis of clinically relevant samples. [Copyright &y& Elsevier]

Abstract: The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A and S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity. [Copyright &y& Elsevier]

Abstract: Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. Studies on VSV assembly led to the creation of a recombinant VSV in which the glycoprotein (G) gene was deleted. This recombinant (rVSV-ΔG) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment; however, because the infectivity of rVSV-ΔG pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-ΔG pseudotypes have facilitated the analysis of virus entry for numerous viral pathogens without the need for specialized containment facilities. The pseudotypes also provide a robust platform to screen libraries for entry inhibitors and to evaluate the neutralizing antibody responses following vaccination. This manuscript describes methods to produce and titer rVSV-ΔG pseudotypes. Procedures to generate rVSV-ΔG stocks and to quantify virus infectivity are also described. These protocols should allow any laboratory knowledgeable in general virological and cell culture techniques to produce successfully replication-restricted rVSV-ΔG pseudotypes for subsequent analysis. [ABSTRACT FROM AUTHOR]

Abstract: The envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) mediate viral entry and are also the primary target of neutralizing antibodies. The gp160 envelope glycoprotein precursor undergoes proteolytic cleavage in the Golgi complex to produce the gp120 exterior glycoprotein and the gp41 transmembrane glycoprotein, which remain associated non-covalently in the trimeric Env complex. Monomeric soluble gp120 has been used extensively to investigate conformational states, structure, antigenicity and immunogenicity of the HIV-1 Env glycoproteins. Expression of gp120 alone (without gp41) leads to the accumulation not only of monomeric gp120 but also an aberrant dimeric form. The gp120 dimers were sensitive to reducing agents. The formation of gp120 dimers was disrupted by a single amino acid change in the inner domain, and was reduced by removal of the V1/V2 variable loops or the N and C termini. Epitopes on the gp120 inner domain and the chemokine receptor-binding surface were altered or occluded by gp120 dimerization. Awareness of the existence and properties of gp120 dimers should assist interpretation of studies of this key viral protein. [Copyright &y& Elsevier]

Abstract: The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at eliciting humoral immunity. Env''s extensive and heterogeneous N-linked glycosylation affects folding, binding to lectin receptors, antigenicity and immunogenicity. We characterized recombinant Env proteins and virus particles produced in mammalian cells that lack N-acetylglucosaminyltransferase I (GnTI), an enzyme necessary for the conversion of oligomannose N-glycans to complex N-glycans. Carbohydrate analyses revealed that trimeric Env produced in GnTI− / − cells contained exclusively oligomannose N-glycans, with incompletely trimmed oligomannose glycans predominating. The folding and conformation of Env proteins was little affected by the manipulation of the glycosylation. Viruses produced in GnTI− / − cells were infectious, indicating that the conversion to complex glycans is not necessary for Env entry function, although virus binding to the C-type lectin DC-SIGN was enhanced. Manipulating Env''s N-glycosylation may be useful for structural and functional studies and for vaccine design. [Copyright &y& Elsevier]

Abstract: Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved nonlinear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection. [Copyright &y& Elsevier]

Abstract: The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP''s overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion. [Copyright &y& Elsevier]

Abstract: The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers. [Copyright &y& Elsevier]