A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU.     

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 use different receptor complexes to enter T cells

Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.     

Similar regulation of cell surface human T-cell leukemia virus type 1 (HTLV-1) surface binding proteins in cells highly and poorly transduced by HTLV-1-pseudotyped virions

Little is known about the requirements for human T-cell leukemia virus type 1 (HTLV-1) entry, including the identity of the cellular receptor(s). Previous studies have shown that although the HTLV receptor(s) are widely expressed on cell lines of various cell types from different species, cell lines differ dramatically in their susceptibility to HTLV-Env-mediated fusion. Human cells (293, HeLa, and primary CD4(+) T cells) showed higher levels of binding at saturation than rodent (NIH 3T3 and NRK) cells to an HTLV-1 SU immunoadhesin. A direct comparison of the binding of the HTLV-1 surface glycoprotein (SU) immunoadhesin and transduction by HTLV-1 pseudotyped virus revealed parallels between the level of binding and the titer for various cell lines. When cells were treated with phorbol myristate acetate (PMA), which down-modulates a number of cell surface molecules, the level of SU binding was markedly reduced. However, PMA treatment only slightly reduced the titer of murine leukemia virus(HTLV-1) on both highly susceptible and poorly susceptible cells. Treatment of target cells with trypsin greatly reduced binding, indicating that the majority of HTLV SU binding is to proteins. Polycations, which enhance the infectivity of several other retroviruses, inhibited HTLV-1 Env-mediated binding and entry on both human and rodent cells. These results suggest that factors other than the number of primary binding receptors are responsible for the differences in the titers of HTLV-1 pseudotypes between highly susceptible cells and poorly susceptible cells.     

Human T-cell leukemia virus type 1 receptor expression among syncytium-resistant cell lines revealed by a novel surface glycoprotein-immunoadhesin

The envelope glycoproteins of human T-cell leukemia virus type 1 (HTLV-1) perform functions that are crucial for virus entry into cells. The surface glycoprotein (SU) is responsible for viral recognition of, and binding to, target cells through its interaction with an unknown cell surface receptor. To facilitate molecular analysis of the receptor-binding properties of SU and to characterize the cellular receptor employed by HTLV-1, we have expressed a recombinant SU fused to the Fc domain of human immunoglobulin G. Here, we demonstrate that this novel SU-immunoadhesin retains both the biochemical properties of Fc and the receptor-binding specificity of the HTLV-1 SU. We use this SU-immunoadhesin to demonstrate, by direct cell surface binding assays, that the receptor used by HTLV-1 has been conserved through vertebrate evolution. Moreover, using murine-human somatic cell hybrids we provide data that do not support the previously assigned location for the HTLV-1 receptor on human chromosome 17. Most importantly, we show that many cell lines that are resistant to HTLV-1 envelope-mediated infection and syncytium formation express functional receptors that are recognized by the HTLV-1 SU. Based on our results, we suggest that for some HTLV-1-resistant cell lines the block to viral entry occurs at a late post-receptor-binding step of the entry process. Our findings will be of value in developing new strategies to identify the cellular receptor used by HTLV-1.     

Bovine leukemia virus SU protein interacts with zinc,and mutations within two interacting regions differently affect viral fusion and infectivity in vivo

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.     

Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.     

Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.     

Analysis and function of prototype foamy virus envelope N glycosylation

The prototype foamy virus (PFV) glycoprotein, which is essential for PFV particle release, displays a highly unusual biosynthesis, resulting in posttranslational cleavage of the precursor protein into three particle-associated subunits, i.e., leader peptide (LP), surface (SU), and transmembrane (TM). Glycosidase digestion of metabolically labeled PFV particles revealed the presence of N-linked carbohydrates on all subunits. The differential sensitivity to specific glycosidases indicated that all oligosaccharides on LP and TM are of the high-mannose or hybrid type, whereas most of those attached to SU, which contribute to about 50% of its molecular weight, are of the complex type. Individual inactivation of all 15 potential N-glycosylation sites in PFV Env demonstrated that 14 are used, i.e., 1 out of 2 in LP, 10 in SU, and 3 in TM. Analysis of the individual altered glycoproteins revealed defects in intracellular processing, support of particle release, and infectivity for three mutants, having the evolutionarily conserved glycosylation sites N8 in SU or N13 and N15 in the cysteine-rich central "sheets-and-loops" region of TM inactivated. Examination of alternative mutants with mutations affecting glycosylation or surrounding sequences at these sites indicated that inhibition of glycosylation at N8 and N13 most likely is responsible for the observed replication defects, whereas for N15 surrounding sequences seem to contribute to a temperature-sensitive phenotype. Taken together these data demonstrate that PFV Env and in particular the SU subunit are heavily N glycosylated and suggest that although most carbohydrates are dispensable individually, some evolutionarily conserved sites are important for normal Env function of FV isolates from different species.     

The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events.

To examine the contribution of the transmembrane envelope glycoprotein (TM) to the infectivity of the human T-cell leukemia virus type 1 (HTLV-1), single amino acid substitutions were introduced throughout its ectodomain. The mutated envelopes were tested for intracellular maturation and for functions, including ability to elicit syncytium formation and ability to mediate cell-to-cell transmission of the virus. Three major phenotypes, defining three functionally distinct regions, were identified. (i) Mutations causing defects in intracellular maturation of the envelope precursor are mostly distributed in the central portion of the TM ectodomain, containing the immunosuppressive peptide. This region, which includes vicinal cysteines thought to form an intramolecular disulfide bridge, is probably essential for correct folding of the protein. (ii) Mutations resulting in reduced syncytium-forming ability despite correct intracellular maturation are clustered in the amino-terminal part of the TM ectodomain, within the leucine zipper-like motif. Similar motifs with a propensity to form coiled-coil structures have been implicated in the fusion process driven by other viral envelope proteins, and HTLV-1 may thus conform to this general rule for viral fusion. (iii) Mutants with increased syncytium-forming ability define a region immediately amino-terminal to the membrane-spanning domain. Surprisingly, these mutants exhibited severe defects in infectivity, despite competence for fusion. Existence of this phenotype indicates that capacity for cell-to-cell fusion is not sufficient to ensure viral entry, even in cell-to-cell transmission. The ectodomain of the TM glycoprotein thus may be involved in postfusion events required for full infectivity of HTLV-1, which perhaps represents a unique feature of this poorly infectious retrovirus.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

Activation of membrane fusion by murine leukemia viruses is controlled in cis or in trans by interactions between the receptor-binding domain and a conserved disulfide loop of the carboxy terminus of the surface glycoprotein

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in trans by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in cis, via the proper RBD of the viral Env itself, or in trans, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers.     

B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.     

Epidemiological Analysis of HTLV-1 and HTLV-2 Infection among Different Population in Central China

Background

HTLV-1 and HTLV-2 are retroviruses linked etiologically to various human diseases, and both of them can be transmitted by vertical route, sexual intercourse, blood transfusion and intravenous drug use. Recently, some HTLV-infected cases have been reported and this virus is mainly present in the Southeast coastal areas in China, but has not been studied for the people in Central China.

Objectives

To know the epidemiologic patterns among different population samples in Central China and further identify risk factor for HTLV-1 and HTLV-2 infection.

Methods

From January 2008 to December 2011, 5480 blood samples were screened for HTLV-1/2 antibodies by using enzyme immunoassay, followed by Western Blot.

Results

The prevalence of HTLV-1 and HTLV-2 was found with infection rates 0.13% and 0.05% among all population samples for HTLV-1 and HTLV-2, respectively. The highest percentages of infection, 0.39% and 0.20%, were found in the high risk group, while only 0.06% and 0.03% in the blood donor group. There was only one case of HTLV-1 infection (0.11%) among patients with malignant hematological diseases. Of seven HTLV-1 positive cases, six were co-infected with HBV, two with HCV and one with HIV. Among three HTLV-2 positive individuals all were co-infected with HBV, one with HCV.

Conclusions

HTLV-1 and HTLV-2 have been detected in the Central China at low prevalence, with the higher infection rate among high risk group. It was also found that co-infection of HTLV-1/2 with HIV and HBV occurred, presumably due to their similar transmission routes. HTLV-1/2 antibody screen among certain population would be important to prevent the spread of the viruses.     

Transmembrane domains of hepatitis C virus envelope glycoproteins: residues involved in E1E2 heterodimerization and involvement of these domains in virus entry

The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins.     

Cytopathic Mechanisms of HIV-1

The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein.     

Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.     

Definition of an amino-terminal domain of the human T-cell leukemia virus type 1 envelope surface unit that extends the fusogenic range of an ecotropic murine leukemia virus

Murine leukemia viruses (MuLV) and human T-cell leukemia viruses (HTLV) are phylogenetically highly divergent retroviruses with distinct envelope fusion properties. The MuLV envelope glycoprotein surface unit (SU) comprises a receptor-binding domain followed by a proline-rich region which modulates envelope conformational changes and fusogenicity. In contrast, the receptor-binding domain and SU organization of HTLV are undefined. Here, we describe an HTLV/MuLV envelope chimera in which the receptor-binding domain and proline-rich region of the ecotropic MuLV were replaced with the potentially corresponding domains of the HTLV-1 SU. This chimeric HTLV/MuLV envelope was processed, specifically interfered with HTLV-1 envelope-mediated fusion, and similar to MuLV envelopes, required cleavage of its cytoplasmic tail to exert significant fusogenic properties. Furthermore, the HTLV domain defined here broadened ecotropic MuLV envelope-induced fusion to human and simian cell lines.