Second site reversion of HIV-1 envelope protein baseplate mutations maps to the matrix protein.

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Domain Organization of Lentiviral and Betaretroviral Surface Envelope Glycoproteins Modeled with AlphaFold.

The surface envelope glycoproteins of nonprimate lentiviruses and betaretroviruses share sequence similarity with the inner proximal domain ß-sandwich of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein that faces the transmembrane glycoprotein as well as patterns of cysteine and glycosylation site distribution that points to a similar two-domain organization in at least some lentiviruses. Here, high-reliability models of the surface glycoproteins obtained with the AlphaFold algorithm are presented for the gp135 glycoprotein of the small ruminant caprine arthritis-encephalitis (CAEV) and visna lentiviruses and the betaretroviruses Jaagsiekte sheep retrovirus (JSRV), mouse mammary tumor virus (MMTV), and consensus human endogenous retrovirus type K (HERV-K). The models confirm and extend the inner domain structural conservation in these viruses and identify two outer domains with a putative receptor binding site in the CAEV and visna virus gp135. The location of that site is consistent with patterns of sequence conservation and glycosylation site distribution in gp135. In contrast, a single domain is modeled for the JSRV, MMTV, and HERV-K betaretrovirus envelope proteins that is highly conserved structurally in the proximal region and structurally diverse in apical regions likely to interact with cell receptors. The models presented here identify sites in small ruminant lentivirus and betaretrovirus envelope glycoproteins likely to be critical for virus entry and virus neutralization by antibodies and will facilitate their functional and structural characterization. IMPORTANCE Structural information on the surface envelope proteins of lentiviruses and related betaretroviruses is critical to understand mechanisms of virus-host interactions. However, experimental determination of these structures has been challenging, and only the structure of the human immunodeficiency virus type 1 gp120 has been determined. The advent of the AlphaFold artificial intelligence method for structure prediction allows high-quality modeling of the structures of small ruminant lentiviral and betaretroviral surface envelope proteins. The models are consistent with much of the previously described experimental data, show regions likely to interact with receptors, and identify domains that may be involved in mechanisms of antibody neutralization resistance in the small ruminant lentiviruses. The models will allow more precise design of mutants to further determine mechanisms of viral entry and immune evasion in this group of viruses and constructs for structural determination of these surface envelope proteins.

Global Increases in Human Immunodeficiency Virus Neutralization Sensitivity Due to Alterations in the Membrane-Proximal External Region of the Envelope Glycoprotein Can Be Minimized by Distant State 1-Stabilizing Changes.

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ([gp120/gp41]3) into more "open" conformations. HIV-1 Env on the viral membrane is maintained in a State-1 conformation that resists binding and neutralization by commonly elicited antibodies. Premature triggering of Env before the virus engages a target cell typically leads to increased susceptibility to spontaneous inactivation or ligand-induced neutralization. Here, we showed that single amino acid substitutions in the gp41 membrane-proximal external region (MPER) of a primary HIV-1 strain resulted in viral phenotypes indicative of premature triggering of Env to downstream conformations. Specifically, the MPER changes reduced viral infectivity and globally increased virus sensitivity to poorly neutralizing antibodies, soluble CD4, a CD4-mimetic compound, and exposure to cold. In contrast, the MPER mutants exhibited decreased sensitivity to the State 1-preferring inhibitor, BMS-806, and to the PGT151 broadly neutralizing antibody. Depletion of cholesterol from virus particles did not produce the same State 1-destabilizing phenotypes as MPER alterations. Notably, State 1-stabilizing changes in Env distant from the MPER could minimize the phenotypic effects of MPER alteration but did not affect virus sensitivity to cholesterol depletion. Thus, membrane-proximal gp41 elements contribute to the maintenance of the pretriggered Env conformation. The conformationally disruptive effects of MPER changes can be minimized by distant State 1-stabilizing Env modifications, a strategy that may be useful in preserving the native pretriggered state of Env. IMPORTANCE The pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult. One reason is that the pretriggered shape of Env is unstable and dependent on interactions near the viral membrane. Here, we showed that the membrane-proximal external region (MPER) of Env plays an important role in maintaining Env in a pretriggered shape. Alterations in the MPER resulted in global changes in Env conformation that disrupted its pretriggered shape. We also found that these disruptive effects of MPER changes could be minimized by distant Env modifications that stabilized the pretriggered shape. These modifications may be useful for preserving the native shape of Env for structural and vaccine studies.

Deep-Time Structural Evolution of Retroviral and Filoviral Surface Envelope Proteins.

The retroviral surface envelope protein subunit (SU) mediates receptor binding and triggers membrane fusion by the transmembrane (TM) subunit. SU evolves rapidly under strong selective conditions, resulting in seemingly unrelated SU structures in highly divergent retroviruses. Structural modeling of the SUs of several retroviruses and related endogenous retroviral elements with AlphaFold 2 identifies a TM-proximal SU ß-sandwich structure that has been conserved in the orthoretroviruses for at least 110 million years. The SU of orthoretroviruses diversified by the differential expansion of the ß-sandwich core to form domains involved in virus-host interactions. The ß-sandwich domain is also conserved in the SU equivalent GP1 of Ebola virus although with a significantly different orientation in the trimeric envelope protein structure relative to the ß-sandwich of human immunodeficiency virus type 1 gp120, with significant evidence for divergent rather than convergent evolution. The unified structural view of orthoretroviral SU and filoviral GP1 identifies an ancient, structurally conserved, and evolvable domain underlying the structural diversity of orthoretroviral SU and filoviral GP1. IMPORTANCE The structural relationships of SUs of retroviral groups are obscured by the high rate of sequence change of SU and the deep-time divergence of retroviral lineages. Previous data showed no structural or functional relationships between the SUs of type C gammaretroviruses and lentiviruses. A deeper understanding of structural relationships between the SUs of different retroviral lineages would allow the generalization of critical processes mediated by these proteins in host cell infection. Modeling of SUs with AlphaFold 2 reveals a conserved core domain underlying the structural diversity of orthoretroviral SUs. Definition of the conserved SU structural core allowed the identification of a homologue structure in the SU equivalent GP1 of filoviruses that most likely shares an origin, unifying the SU of orthoretroviruses and GP1 of filoviruses into a single protein family. These findings will allow an understanding of the structural basis for receptor-mediated membrane fusion mechanisms in a broad range of biomedically important retroviruses.

Reconstitution of Fusion-Competent Human Placental Fusogen Syncytin-2.

Mammalian placenta formation requires continuous fusion of trophoblasts. Human endogenous retrovirus-derived proteins syncytin-1 and syncytin-2 mediate cell-cell fusion of placental cytotrophoblasts to form syncytiotrophoblasts in primates, which is required for normal placenta function and fetal development. Syncytins are post-translationally cleaved by the endoprotease furin into surface (SU) and transmembrane (TM) subunits for activation. Little is currently known about the molecular mechanisms of syncytin-mediated cell-cell fusion, and their functions have not been well studied in vitro. Here, we express tagged syncytin-2 in mammalian HEK293T cells and demonstrate that the tagging greatly influences the cleavage and fusogenic activity of syncytin-2. By detecting the N-terminal tagged SU, we find that it is released into the extracellular space during the fusion process. Furthermore, when N-linked glycosylation and disulfide bond formation are blocked, the cleavage and fusogenic activity of syncytin-2 are inhibited. Finally, we were able to purify functional syncytin-2 from HEK293T cells and incorporate it into proteoliposomes. These findings lay a solid foundation for interogating the molecular mechanisms of syncytin-2-mediated cell-cell fusion in vitro.

An ancient retroviral RNA element hidden in mammalian genomes and its involvement in co-opted retroviral gene regulation.

BACKGROUND: Retroviruses utilize multiple unique RNA elements to control RNA processing and translation. However, it is unclear what functional RNA elements are present in endogenous retroviruses (ERVs). Gene co-option from ERVs sometimes entails the conservation of viral cis-elements required for gene expression, which might reveal the RNA regulation in ERVs. RESULTS: Here, we characterized an RNA element found in ERVs consisting of three specific sequence motifs, called SPRE. The SPRE-like elements were found in different ERV families but not in any exogenous viral sequences examined. We observed more than a thousand of copies of the SPRE-like elements in several mammalian genomes; in human and marmoset genomes, they overlapped with lineage-specific ERVs. SPRE was originally found in human syncytin-1 and syncytin-2. Indeed, several mammalian syncytin genes: mac-syncytin-3 of macaque, syncytin-Ten1 of tenrec, and syncytin-Car1 of Carnivora, contained the SPRE-like elements. A reporter assay revealed that the enhancement of gene expression by SPRE depended on the reporter genes. Mutation of SPRE impaired the wild-type syncytin-2 expression while the same mutation did not affect codon-optimized syncytin-2, suggesting that SPRE activity depends on the coding sequence. CONCLUSIONS: These results indicate multiple independent invasions of various mammalian genomes by retroviruses harboring SPRE-like elements. Functional SPRE-like elements are found in several syncytin genes derived from these retroviruses. This element may facilitate the expression of viral genes, which were suppressed due to inefficient codon frequency or repressive elements within the coding sequences. These findings provide new insights into the long-term evolution of RNA elements and molecular mechanisms of gene expression in retroviruses.

Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins.

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Insights into the trimeric HIV-1 envelope glycoprotein structure.

The HIV-1 envelope glycoprotein (Env) trimer is responsible for receptor recognition and viral fusion with CD4(+) T cells, and is the sole target for neutralizing antibodies. Thus, understanding its molecular architecture is of significant interest. However, the Env trimer has proved to be a challenging target for 3D structure determination. Recent electron microscopy (EM) and X-ray structures have at last enabled us to decipher the structural complexity and unique features of the Env trimer, and how it is recognized by an ever-expanding arsenal of potent broadly neutralizing antibodies. We describe our current knowledge of the Env trimer structure in the context of exciting recent developments in the identification and characterization of HIV broadly neutralizing antibodies.

Design and characterization of a self-assembling protein nanoparticle displaying HIV-1 Env V1V2 loop in a native-like trimeric conformation as vaccine antigen.

The RV144 HIV-1 clinical trial demonstrated modest vaccine efficacy and identified IgG antibodies against the Env V1V2 loop that inversely correlated with risk of infection. Based upon these results, we chose the Self-Assembling Protein Nanoparticle platform to present the V1V2 loop in a native-like conformation. We hypothesized this approach would lead to generation of conformation-specific IgG antibodies to V1V2. Our vaccine, V1V2-SHB-SAPN, was designed to present twenty copies of the V1V2 trimer. Particles were characterized for size, shape, and binding to monoclonal antibodies that recognize the V2 and V1V2 loops. Immunization induced IgG antibodies to V1, V2, V1V2 and to gp70V1V2 (AE/A244) capture antigens in mice. The presence of the Army Liposome Formulation induced a four-fold increase in IgG titers to gp70V1V2 and the adjuvanted V1V2-SHB-SAPN group had statistically higher IgG titers than sequence- and dose-matched V1V2 peptide controls. In conclusion, V1V2-SHB-SAPN vaccine presented the V1V2 loop in native-like conformation, as indicated by PGT145 binding, and induced high titers of IgG antibodies.

A novel neuropilin-1-binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 µM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Comprehensive antigenic map of a cleaved soluble HIV-1 envelope trimer.

The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.

Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure.

The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAVL) in donkeys (EIAVD) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAVL) with attenuated strains serially passaged in donkey MDM (EIAVDLV) and donkey dermal cells (EIAVFDDV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAVFDDV infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAVFDDV. Results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum neutralizing antibodies that were also able to recognize parental viruses. This study indicates that these mutations played an important role in the attenuation of the EIAV vaccine strains.

Detection of Jaagsiekte sheep retrovirus in the peripheral blood during the pre-clinical period of ovine pulmonary adenomatosis.

The envelope protein (Env) of the Jaagsiekte sheep retrovirus (JSRV) is known to be a unique oncoprotein responsible for inducing ovine pulmonary adenocarcinoma (OPA). The objective of this study was to prepare a specific monoclonal antibody (mAb) against the JSRV Env protein using bioinformatic analysis. According to the structure and epitope prediction results of JSRV Env, the JSRV-Env572-615 antigen was prepared via peptide synthesis (amino acid sequence 572-615, denoted as JSRV-Env572-615). BALB/c mice were immunized to prepare the anti-JSRV-Env572-615 mAb. Spleen cells were fused with SP2/0 myeloma cells after being screened by indirect ELISA and cloned by limiting dilution. The specificity of mAb was evaluated by western blot analysis and immunohistochemistry assays. Western blot results showed that the JSRV Env protein was able to bind to mAb with high specificity. Immunohistochemistry assays demonstrated that the mAb was able to recognize JSRV Env in adenomatous hyperplasia of the lung. Furthermore, JSRV was detected in peripheral blood leukocytes during the pre-clinical period of OPA in 2 of the 25 sheep using this newly synthesized mAb. Therefore, this mAb may be a useful tool for the detection of JSRV in sheep.

Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA.

Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.

HIV-1 Superinfection Resembles Primary Infection.

The relevance of superinfection as a model to identify correlates of protection against human immunodeficiency virus (HIV) depends on whether the superinfecting transmission resembles primary infection, which has not been established. Here, we characterize the genetic bottleneck in superinfected individuals for the first time. In all 3 cases, superinfection produced a spike in viral load and could be traced to a single, C-C chemokine receptor 5-tropic founder virus with shorter, less glycosylated variable regions than matched chronic viruses. These features are consistent with primary HIV transmission and provide support for the use of superinfection as a model to address correlates of protection against HIV.

A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

One hundred twenty years of koala retrovirus evolution determined from museum skins.

Although endogenous retroviruses are common across vertebrate genomes, the koala retrovirus (KoRV) is the only retrovirus known to be currently invading the germ line of its host. KoRV is believed to have first infected koalas in northern Australia less than two centuries ago. We examined KoRV in 28 koala museum skins collected in the late 19th and 20th centuries and deep sequenced the complete proviral envelope region from five northern Australian specimens. Strikingly, KoRV env sequences were conserved among koalas collected over the span of a century, and two functional motifs that affect viral infectivity were fixed across the museum koala specimens. We detected only 20 env polymorphisms among the koalas, likely representing derived mutations subject to purifying selection. Among northern Australian koalas, KoRV was already ubiquitous by the late 19th century, suggesting that KoRV evolved and spread among koala populations more slowly than previously believed. Given that museum and modern koalas share nearly identical KoRV sequences, it is likely that koala populations, for more than a century, have experienced increased susceptibility to diseases caused by viral pathogenesis.