Lactic acid bacterial surface display of scytovirin inhibitors for anti-ebolavirus infection.

Scytovirin (SVN) is a lectin from cyanobacteria which has a strong inhibitory activity against Ebola virus infection. We engineered scytovirin as the inhibitor for surface display of lactic acid bacteria to block Ebola virus infection. Two different bacterial strains (Lactobacillus casei and Lactococcus lactis) were successfully engineered for scytovirin expression on the bacterial surface. These bacteria were found to be effective at neutralizing pseudotyped Ebolavirus in a cell-based assay. This approach can be utilized for prophylactic prevention, as well as for treatment. Since lactic acid bacteria can colonize the human body, a long-term efficacy could be achieved. Furthermore, this approach is also simple and cost-effective and can be easily applied in the regions of Ebola outbreaks in the developing countries.

Displaying and delivering viral membrane antigens via WW domain-activated extracellular vesicles.

Membrane proteins expressed on the surface of enveloped viruses are conformational antigens readily recognized by B cells of the immune system. An effective vaccine would require the synthesis and delivery of these native conformational antigens in lipid membranes that preserve specific epitope structures. We have created an extracellular vesicle-based technology that allows viral membrane antigens to be selectively recruited onto the surface of WW domain-activated extracellular vesicles (WAEVs). Budding of WAEVs requires secretory carrier-associated membrane protein 3, which through its proline-proline-alanine-tyrosine motif interacts with WW domains to recruit fused viral membrane antigens onto WAEVs. Immunization with influenza and HIV viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal viral infection. WAEVs thus represent a versatile platform for presenting and delivering membrane antigens as vaccines against influenza, HIV, and potentially many other viral pathogens.

Ligand-based design of peptide entry inhibitors targeting the endosomal receptor binding site of filoviruses.

Filoviruses enter cells through macropinocytosis and trafficking into the endosomes in which they bind to the receptor Niemann-Pick C1 protein (NPC1) for membrane fusion and entry into the cytoplasm. The endosomal receptor-binding is critical step for filovirus entry. Designing inhibitors to block receptor binding will prevent viral entry. Using available binding structural information from the co-crystal structures of the viral GP with the receptor NPC1 or with monoclonal antibodies, we have conducted structure-based design of peptide inhibitors to target the receptor binding site (RBS). The designed peptides were tested for their inhibition activity against pseudo-typed or replication-competent viruses in a cell-based assay. The results indicate that these peptides exhibited strong activities against both Ebola and Marburg virus infection. It is expected that these peptides can be further developed for therapeutic use to treat filovirus infection and combat the outbreaks.

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

Identification of filovirus entry inhibitors targeting the endosomal receptor NPC1 binding site.

Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC50 value (0.86 µM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC50 value was about 100 µM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection.

Mandibulofacial Dysostosis Attributed to a Recessive Mutation of CYP26C1 in Hereford Cattle.

In spring 2020, six Hereford calves presented with congenital facial deformities attributed to a condition we termed mandibulofacial dysostosis (MD). Affected calves shared hallmark features of a variably shortened and/or asymmetric lower mandible and bilateral skin tags present 2-10 cm caudal to the commissure of the lips. Pedigree analysis revealed a single common ancestor shared by the sire and dam of each affected calf. Whole-genome sequencing (WGS) of 20 animals led to the discovery of a variant (Chr26 g. 14404993T>C) in Exon 3 of CYP26C1 associated with MD. This missense mutation (p.L188P), is located in an α helix of the protein, which the identified amino acid substitution is predicted to break. The implication of this mutation was further validated through genotyping 2 additional affected calves, 760 other Herefords, and by evaluation of available WGS data from over 2500 other individuals. Only the affected individuals were homozygous for the variant and all heterozygotes had at least one pedigree tie to the suspect founder. CYP26C1 plays a vital role in tissue-specific regulation of retinoic acid (RA) during embryonic development. Dysregulation of RA can result in teratogenesis by altering the endothelin-1 signaling pathway affecting the expression of Dlx genes, critical to mandibulofacial development. We postulate that this recessive missense mutation in CYP26C1 impacts the catalytic activity of the encoded enzyme, leading to excess RA resulting in the observed MD phenotype.

Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that localize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection.

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-α (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer+ T cells were inconsistently detected, the ß1-adrenergic receptor 181-200/211-230 or branched chain α-ketoacid dehydrogenase kinase 111-130 dextramer+ cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections.

Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681-700, VP1 721-740 and VP1 771-790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721-740 and VP1 681-700 followed by VP1 771-790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721-740 and VP1 681-700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

Blocking HIV-1 Infection by Chromosomal Integrative Expression of Human CD4 on the Surface of Lactobacillus acidophilus ATCC 4356.

Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study was to employ these lactobacilli to combat human immunodeficiency virus type 1 (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered Lactobacillus acidophilus ATCC 4356 to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different subtypes and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission.IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus bacteria stably expressing the HIV-1 receptor CD4 to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins, such as CD4, following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.

Nucleolin Is a Functional Binding Protein for Salinomycin in Neuroblastoma Stem Cells.

The aim of this study is to illuminate a novel therapeutic approach by identifying a functional binding target of salinomycin, an emerging anticancer stem cell (CSC) agent, and to help dissect the underlying action mechanisms. By utilizing integrated strategies, we identify that nucleolin (NCL) is likely a salinomycin-binding target and a critical regulator involved in human neuroblastoma (NB) CSC activity. Salinomycin markedly suppresses NB CD34 expression and reduces CD34+ cell population in an NCL-dependent manner via disruption of the interaction of NCL with CD34 promoter. The elevated levels of NCL expression in NB tumors are associated with poor patient survival. Altogether, these results indicate that NCL is likely a novel functional salinomycin-binding target that exhibits the potential to be a prognostic marker for NB therapy.

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association.

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

Contribution of the gp120 V3 loop to envelope glycoprotein trimer stability in primate immunodeficiency viruses.

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Env-mediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses.

Characterization of twin-cysteine motif in the V2-loop region of gp120 in primate lentiviruses.

The twin-cysteine motif (TCM) in the V2 loop region of gp120, identified in our previous report on the simian immunodeficiency virus mac239 (SIVmac239), is a conserved evolutionary element in all primate lentiviruses except for HIV-1 which has lost the TCM during cross-species transmission. In this study, we have further explored the TCM in other SIV and HIV-2 strains. Our data shows that strains from different evolutionary lineages have different phenotypes when the twin-cysteines are removed. In the SIVsm/HIV-2 lineage, removal of the twin-cysteines decreases envelope trimer stability, but in the SIVagm lineage, a blockage of gp160 processing is observed. Molecular modeling has confirmed that the twin-cysteines do form a disulfide bond in the gp120 subunit, which interacts with the V1 loop to stabilize the envelope trimer. Therefore, we hypothesize that if the TCM is added back to HIV-1, it will enhance envelope stability for vaccine immunogen design.

Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.

Persistence of SIV in the brain of SIV-infected Chinese rhesus macaques with or without antiretroviral therapy.

Persistence of HIV-1 reservoirs in the central nervous system (CNS) is an obstacle to cure strategies. However, little is known about residual viral distribution, viral replication levels, and genetic diversity in different brain regions of HIV-infected individuals on combination antiretroviral therapy (cART). Because myeloid cells particularly microglia are likely major reservoirs in the brain, and more microglia exist in white matter than gray matter in a human brain, we hypothesized the major viral reservoirs in the brain are the white matter reflected by higher levels of viral DNA. To address the issue, we used the Chinese rhesus macaque (ChRM) model of SIV infection, and treated 11 SIVmac251-infected animals including long-term nonprogressors with cART for up to 24 weeks. SIV reservoirs were assessed by SIV DNA levels in 16 specific regions of the brain and 4 regions of spinal cord. We found relatively high frequencies of SIV in basal ganglia and brain stem compared to other regions. cART-receiving animals had significantly lower SIV DNA levels in the gray matter than white matter. Moreover, a shortened envelope gp120 with 21 nucleotide deletions and guanine-to-adenine hypermutations were observed. These results demonstrate that SIV enters the CNS in SIV-infected ChRM with a major reservoir in the white matter after cART; the SIV/ChRM/cART is an appropriate model for studying HIV CNS reservoirs and testing new eradication strategies. Further, examining multiple regions of the CNS may be needed when assessing whether an agent is successful in reducing the size of SIV reservoirs in the CNS.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Tryptophan 375 stabilizes the outer-domain core of gp120 for HIV vaccine immunogen design.

The outer-domain core of gp120 may serve as a better HIV vaccine immunogen than the full-length gp120 because of its greater stability and immunogenicity. In our previous report, we introduced two disulfide bonds to the outer-domain core of gp120 to fix its conformation into a CD4-bound state, which resulted in a significant increase in its immunogenicity when compared to the wild-type outer-domain core. In this report, to further improve the immunogenicity of the outer-domain core based immunogen, we have introduced a Tryptophan residue at gp120 amino acid sequence position 375 (375S/W). Our data from immunized guinea pigs indeed shows a striking increase in the immune response due to this stabilized core outer-domain. Therefore, we conclude that the addition of 375W to the outer-domain core of gp120 further stabilizes the structure of immunogen and increases the immunogenicity.

Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function.

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1 and the fusion-mediating GP2 subunits and incorporated into virions to initiate infection. GP1 and GP2 form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1 NGSs are not critical, the two GP2 NGSs, Asn563 and Asn618, are essential for GP function. Further analysis uncovered that Asn563 and Asn618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn563 and Asn618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.

Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus.

Binding of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological layers (layer 1, layer 2, and layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved tryptophan at position 375 (Trp 375) in HIV-2/SIVsmm. In this study, we investigated the role of SIV layer 3 in viral entry, cell-to-cell fusion, and CD4 binding. We observed that a network of interactions involving some residues of the ß8-α5 region in SIVmac239 layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity, which directly contacts CD4. In summary, our results suggest that layer 3 in SIV has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in layer 3 from HIV-1 and that from SIV. IMPORTANCE: CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of layers 1 to 3 for HIV-1 and layers 1 and 2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV layer 3 remains unknown. Here we report that SIV layer 3 has a greater impact on CD4 binding than does layer 3 in HIV-1 gp120. This work defines lineage-specific differences in layer 3 from HIV-1 and SIV.

Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor.

Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor (CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibitors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an HIV-1 entry inhibitor.