Envelope-receptor Interactions Research Articles

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

An in silico approach for identification of novel inhibitors as a potential therapeutics targeting HIV-1 viral infectivity factor.

Currently available antiviral drugs target the pol-encoded retroviral enzymes or integrases, in addition, inhibitors that target HIV-1 envelope-receptor interactions have also been recently approved. Recent understanding of the interactions between HIV-1 and host restriction factors has provided fresh avenues for development of novel antiviral drugs. For example, viral infectivity factor (Vif) now surfaced as an important therapeutic target in treatment of HIV infection. Vif suppresses A3G antiviral activity by targeting these proteins for polyubiquitination and proteasomal degradation. In the present study we analyzed the inhibitory potential of VEC5 and RN18 to inhibit the Vif-A3G interaction through protein- protein docking studies. Perusal of the study showed that, VEC5 and RN18 though inhibits the interaction however showed sub optimal potential. To overcome this set back, we identified 35 structural analogues of VEC5 and 18 analogues of RN18 through virtual screening approach. Analogue with PubCID 71624757 and 55358204 (AKOS006479723) -structurally akin to VEC5 and RN18 respectively showed much appreciable interaction than their respective parent compound. Evident from Vif-A3G; protein - protein docking studies, analogue PubCID 71624757 demonstrated 1.08 folds better inhibitory potential than its parent compound VEC5 while analogue PubCID 55358204 was 1.15 folds better than RN18. Further these analogues passed drug likeness filters and predicted to be non- toxic. We expect these analogues can be put to pharmacodynamic studies that can pave way the breakthrough in HIV therapeutics.

HIV-1 envelope-receptor interactions required for macrophage infection and implications for current HIV-1 cure strategies.

Myeloid cells residing in the CNS and lymphoid tissues are targets for productive HIV-1 replication, and their infection contributes to the pathological manifestations of HIV-1 infection. The Envs can adopt altered configurations to overcome entry restrictions in macrophages via a more efficient and/or altered mechanism of engagement with cellular receptors. This review highlights evidence supporting an important role for macrophages in HIV-1 pathogenesis and persistence, which need to be considered for strategies aimed at achieving a functional or sterilizing cure. We also highlight that the molecular mechanisms underlying HIV-1 tropism for macrophages are complex, involving enhanced and/or altered interactions with CD4, CCR5, and/or CXCR4, and that the nature of these interactions may depend on the anatomical location of the virus.

Primate Gammaretroviruses Require an Ancillary Factor Not Required for Murine Gammaretroviruses To Infect BHK Cells

BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.

Functional murine leukemia virus vectors pseudotyped with the visna virus envelope show expanded visna virus cell tropism.

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.

Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis.

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.

Changes in a Murine Leukemia Virus (MLV) Receptor Encoded by an Alphavirus Vector during Passage in Cells Expressing the MLV Envelope

We constructed alphavirus vectors encoding the ecotropic murine leukemia virus (MLV) receptor mCAT1. Cells electroporated with these vector RNAs expressed mCAT1 protein and fused with cells that expressed a fusogenic form of the MLV envelope on the cell surface. Electroporated cells also released submicron particles that were infectious in envelope-expressing cells. Infection resulted in giant syncytia that could be enumerated by plaque assay. Cell-free supernatants could be serially passaged and contained up to 104 infectious units/ml. To determine whether repeated passage would select for functional variants of the receptor, we analyzed mCAT1 amplified by RT-PCR after 12 serial passages. Several amino acid substitutions were identified that encoded functional receptor variants. In independent experiments, variants containing an isoleucine or a leucine instead of a phenylalanine at position 224 in the third extracellular domain of the receptor arose spontaneously and outgrew the parental vector, indicating that mutations at this site are highly selected in this system. This region of the receptor has previously been implicated in the envelope–receptor interaction. This alphavirus vector system provides a novel method for generating and selecting functional variants of viral receptors

Opposite effects of SDF-1 on human immunodeficiency virus type 1 replication.

The alpha-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.

Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo.

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.

Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1.

We have investigated the cellular uptake of Gag p24 shortly after exposure of cells to human immunodeficiency virus (HIV) particles. In the absence of envelope glycoprotein on virions or of viral receptors or coreceptors at the cell surface, p24 was incorporated in intracellular vesicles but not detected in the cytosolic subcellular fraction. When appropriate envelope-receptor interactions could occur, the nonspecific vesicular uptake was still intense and cytosolic p24 represented 10 to 40% of total intracellular p24. The measurement of cytosolic p24 early after exposure to HIV type 1 is a reliable assay for investigating virus entry and early events leading to authentic cell infection.

Definition of a 14-amino-acid peptide essential for the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor.

Hydrophilic loops in the receptor binding domain of the amphotropic murine leukemia virus (MLV) envelope glycoprotein (SU) are predicted and may participate in SU-receptor interactions. We have replaced five segments of 6 to 15 amino acids located in each of these regions with an 11-amino-acid tag from the vesicular stomatitis virus glycoprotein (VSV-G). Substitution was compatible with envelope processing, transport, and incorporation into virions. However, three substitution mutants showed a temperature-dependent phenotype, suggesting structural unstability. Accessibility of the tagging epitope for a monoclonal anti-VSV-G antibody was greater in oligomeric than in monomeric SUs when insertion was done in VRA, a domain essential for receptor recognition. In contrast, accessibility was independent of structural constraints when insertion was done in VRB, a domain playing an accessory role in receptor binding. Interaction with the amphotropic receptor was investigated by interference assay and study of binding and infection of target cells with MLV particles coated with the substituted envelopes. Envelope-receptor interaction was abolished when substitution was performed in a potential loop-forming segment located at the N-terminal half of VRA. Although interaction was affected to variable extents, depending on the substituted segment, other mutants conserved the ability to interact with the amphotropic receptor. These experiments indicate the 14-amino-acid segment between positions 50 and 64 of SU as an essential determinant of amphotropic-receptor recognition. They also show that a foreign linear epitope can be tolerated in several locations of the amphotropic SU receptor binding site, and this result has implications for the design of targeted retroviral vectors.

Mutational analysis of the envelope protein of spleen necrosis virus

Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding.

We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.

Mapping of receptor binding domains in the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic retrovirus originally isolated from a duck. Although of avian origin, it also replicates on some mammalian cells. SNV-derived retroviral vectors work with high efficiency and have a high potential for various gene transfer applications. However, little is known about the envelope-receptor interactions of this virus. We constructed a series of recombinant envelope proteins to characterize the SU peptide of SNV. We found that, in contrast to the envelope proteins of other retroviruses, truncated envelope proteins of SNV are transported to the cell surface. Surprisingly, particles displaying truncated envelope proteins can still infect cells, although at reduced efficiencies. Furthermore, these proteins can confer partial superinfection interference. Our data suggest that peptides throughout SU are involved in envelope-receptor interactions. To more precisely determine the localization of the main receptor binding domain, point mutations were introduced at certain regions of the SNV SU which are highly conserved among retroviruses belonging to the same receptor interference group. We identified one point mutation in the middle of SU (position 192) which drastically reduced infectivity and strongly reduced the ability to confer superinfection interference. The level of expression was not abolished, and translocation to the cell membrane of the mutant envelope occurred efficiently. This indicates that amino acid 192 may be directly involved in receptor binding.