Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines.

The chemokine receptor CCR5 plays an important role in leukocyte chemotaxis and activation, and also acts as a coreceptor for human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). We provide evidence that CCR5 is O-glycosylated on serine 6 in the NH2 terminus. The O-linked glycans, particularly sialic acid moieties, significantly contribute to binding of the chemokine ligands. By contrast, removal of O-linked oligosaccharide exerted little effect on HIV-1 infection. Sulfation of specific tyrosine residues in the CCR5 NH2 terminus was important for efficient beta-chemokine binding. Thus, as has been observed for the binding of selectins and their ligands, O-linked carbohydrates and tyrosine sulfates play major roles in promoting the interaction of chemokines with CCR5. The resulting flexible arrays of negative charges on the CCR5 surface may allow specific, high-affinity interactions with diverse chemokine ligands. Although this is the first example of O-linked oligosaccharides and tyrosine sulfates playing a role in chemokine binding, the high density of serines, threonines and tyrosines in the N-termini of many CC chemokine receptors suggests that these posttranslational modifications may commonly contribute to chemokine binding.

T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus.

Changes in T cell receptor (TCR) V beta repertoire and their correlation with virologic events were investigated in rhesus monkeys after acute infection with the simian immunodeficiency virus (SIV). 11 genetically defined rhesus monkeys were experimentally infected with SIVmac or a chimeric simian-human immunodeficiency virus (SHIV), and their peripheral blood lymphocytes (PBL) and lymph nodes were prospectively assessed for TCR V beta gene expression. PBL and lymph nodes of the acutely infected monkeys demonstrated an expansion of selected V beta-expressing T lymphocyte subpopulations as early as 3 d after infection. These expanded V beta-expressing lymphocyte subpopulations were comprised predominantly of CD8+ cells. Six of seven infected monkeys sharing a single electrophoretically defined major histocompatibility complex class I allele exhibited a similar expansion of V beta 14-expressing PBL. Sequence analyses of V-D-J segments of TCR-beta cDNA indicated that the V beta-expressing T cell subpopulation expansion can be oligoclonal. SIVmac-specific CD8+ cytotoxic T lymphocytes were demonstrated in both PBL and lymph nodes of the infected monkeys at the time expansion of the selected V beta-expressing cell subpopulations was seen. Finally, the expansion of the selected V beta-expressing lymphocytes in PBL coincided with the emergence and clearance of SIV p27 from the plasma of the infected monkeys. These results demonstrate that acute infection of rhesus monkeys with SIVmac or SHIV results in an expansion of CD8+ lymphocyte subpopulations expressing selected V beta gene families. The selectively expanded T lymphocytes may contribute to early viral clearance after acute SIVmac or SHIV infection.

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4(+) T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

The envelope glycoprotein ectodomains determine the efficiency of CD4+ T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques.

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)-infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian-human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4(+) T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4(+) T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4(+) T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.

The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.

The human immunodeficiency virus-type 1 (HIV-1) envelope glycoproteins interact with receptors on the target cell and mediate virus entry by fusing the viral and cell membranes. The structure of the envelope glycoproteins has evolved to fulfill these functions while evading the neutralizing antibody response. An understanding of the viral strategies for immune evasion should guide attempts to improve the immunogenicity of the HIV-1 envelope glycoproteins and, ultimately, aid in HIV-1 vaccine development.

A transcriptional activator protein encoded by the x-lor region of the human T-cell leukemia virus.

Human T-cell leukemia viruses type I and II (HTLV-I and -II) exhibit several features characteristic of this retroviral family: the presence of an x-lor gene encoding a nuclear protein, transformation properties suggesting the involvement of a virus-associated trans-acting factor, and transcriptional trans-activation of the long terminal repeat (LTR) in infected cells. In the study described here the HTL x-lor products, in the absence of other viral proteins, were able to activate gene expression in trans directed by HTLV LTR. The regulation of the expression of particular genes in trans by HTLV x-lor products suggests that they play a role in viral replication and possibly in transformation of T lymphocytes.

Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells.

The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. Two subtypes of human T lymphotropic virus (HTLV) are associated with different disease phenotypes. In this article it is shown that marked differences exist in the ability of LTR sequences of these subtypes to function as transcriptional elements in differentiated cell types. It is also shown that trans-acting regulatory factors present in HTLV-infected cells stimulate gene expression directed by these LTR sequences in a type-specific manner. These results have implications for understanding the diverse biological effects of HTLV infection.

Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III.

The retrovirus involved in acquired immune deficiency syndrome (HTLV-III/LAV) contains a region that is necessary for stimulation of gene expression directed by the viral long terminal repeat. This region is located between nucleotides 5365 and 5607, immediately 5' to the envelope gene. A doubly-spliced message containing this region could encode an 86-amino acid protein with structural features similar to those of nucleic acid-binding proteins.

Trans activation of the bovine leukemia virus long terminal repeat in BLV-infected cells.

The transcription initiation signals for retroviruses lie within the long terminal repeat (LTR) sequences that flank the integrated provirus. This study shows that factors present in cells infected with bovine leukemia virus (BLV) mediate transcriptional trans activation of the BLV LTR. This phenomenon is similar to that reported for the human T-cell leukemia virus LTR and establishes both structural and functional criteria for inclusion of BLV and human T-cell leukemia viruses in the same family of transforming retroviruses.

Subcellular localization of the product of the long open reading frame of human T-cell leukemia virus type I.

Human T-cell leukemia virus type I (HTLV-I) is a retrovirus associated with adult T-cell leukemia and lymphoma. In addition to containing the gag, pol, and env genes of the chronic leukemia viruses, the genome of HTLV-I contains a long open reading frame (LOR) located between the 3' end of the envelope gene and the 3' long terminal repeat sequence (LTR). It has been suggested that a protein of 42 kilodaltons that is encoded by the LOR region may participate in both trans-acting transcriptional regulation of the viral LTR as well as in the transforming properties of HTLV-I. It is reported here that a significant fraction of the 42-kilodalton HTLV LOR product is located in the nucleus of HTLV-I-infected transformed lymphocytes, a finding that is consistent with its proposed functions.

Structure of 3' terminal region of type II human T lymphotropic virus: evidence for new coding region.

The sequence of the 3' terminus of the human T lymphotropic virus type II (HTLV-II) was determined and compared to the corresponding sequence of HTLV-I. The 1557-nucleotide-long sequence can be divided into a 5' region that is not conserved between the two viruses, and a 3', 1011-nucleotide-long region that is highly conserved and that corresponds precisely with a long open reading frame for both HTLV-I and -II. The proteins that could be encoded by these open reading frames have a molecular weight of about 38,000 and are closely related in primary amino acid sequence. The genomic structure in the 3' region of HTLV was found to be similar to that of bovine leukemia virus.

Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions.

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.

A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding.

The entry of primate immunodeficiency viruses into target cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors, CD4 and members of the chemokine receptor family. The gp120 third variable (V3) loop has been implicated in chemokine receptor binding, but the use of the CCR5 chemokine receptor by diverse primate immunodeficiency viruses suggests the involvement of an additional, conserved gp120 element. Through the use of gp120 mutants, a highly conserved gp120 structure was shown to be critical for CCR5 binding. This structure is located adjacent to the V3 loop and contains neutralization epitopes induced by CD4 binding. This conserved element may be a useful target for pharmacologic or prophylactic intervention in human immunodeficiency virus (HIV) infections.

Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat.

Human T-cell leukemia virus type III (HTLV-III) was recently identified as the probable etiologic agent of the acquired immune deficiency syndrome (AIDS). Here it is shown that, in human T-cell lines infected with HTLV-III, gene expression directed by the long terminal repeat sequence of this virus is stimulated by more than two orders of magnitude compared to matched uninfected cells. The rate of transcription of the HTLV-III long terminal repeat is more than 1000 times that of the SV40 early promoter in one infected cell line. Thus, HTLV-III, like HTLV-I, HTLV-II, and the bovine leukemia virus, is characterized by trans-activation of transcription in infected cells. The efficiency of trans-activation in the case of HTLV-III may account, at least in part, for the virulent nature of HTLV-III infection.

Trans-activator gene of HTLV-II induces IL-2 receptor and IL-2 cellular gene expression.

The human T-lymphotropic viruses types I and II (HTLV-I and -II) have been etiologically linked with certain T-cell leukemias and lymphomas that characteristically display membrane receptors for interleukin-2. The relation of these viruses to this growth factor receptor has remained unexplained. It is demonstrated here that introduction of the trans-activator (tat) gene of HTLV-II into the Jurkat T-lymphoid cell line results in the induction of both interleukin-2 receptor and interleukin-2 gene expression. The coexpression of these cellular genes may play a role in the altering T-cell growth following retroviral infection.

Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III.

Antibodies from the serum of patients with the acquired immune deficiency syndrome (AIDS) or with the AIDS-related complex and from the serum of seropositive healthy homosexuals, recognize two major glycoproteins in cells infected with human T-cell lymphotropic virus type III (HTLV III). These glycoproteins, gp160 and gp120, are encoded by the 2.5-kilobase open reading frame located in the 3' end of the HTLV-III genome, as determined by amino terminus sequence analysis of the radiolabeled forms of these proteins. It is hypothesized that gp160 and gp120 represent the major species of virus-encoded envelope gene products for HTLV-III.

CD4-independent binding of SIV gp120 to rhesus CCR5.

CCR5 and CD4 are coreceptors for immunodeficiency virus entry into target cells. The gp120 envelope glycoprotein from human immunodeficiency virus strain HIV-1(YU2) bound human CCR5 (CCR5hu) or rhesus macaque CCR5 (CCR5rh) only in the presence of CD4. The gp120 from simian immunodeficiency virus strain SIVmac239 bound CCR5rh without CD4, but CCR5hu remained CD4-dependent. The CD4-independent binding of SIVmac239 gp120 depended on a single amino acid, Asp13, in the CCR5rh amino-terminus. Thus, CCR5-binding moieties on the immunodeficiency virus envelope glycoprotein can be generated by interaction with CD4 or by direct interaction with the CCR5 amino-terminus. These results may have implications for the evolution of receptor use among lentiviruses as well as utility in the development of effective intervention.

Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1.

The envelope of the human immunodeficiency virus type 1 (HIV-1) plays a central role in the process of virus entry into the host cell and in the cytopathicity of the virus for lymphocytes bearing the CD4 molecule. Mutations that affect the ability of the envelope glycoprotein to form syncytia in CD4+ cells can be divided into five groups: those that decrease the binding of the envelope protein to the CD4 molecule, those that prevent a post-binding fusion reaction, those that disrupt the anchorage of the envelope glycoprotein in the membrane, those that affect the association of the two subunits of the envelope glycoprotein, and those that affect post-translational proteolytic processing of the envelope precursor protein. These findings provide a functional model of the HIV envelope glycoprotein.

Sequence of the envelope glycoprotein gene of type II human T lymphotropic virus.

The sequence of the envelope glycoprotein gene of type II human T lymphotropic virus (HTLV) is presented. The predicted amino acid sequence is similar to that of the corresponding protein of HTLV type I, in that the proteins share the same amino acids at 336 of 488 residues, and 68 of the 152 differences are of a conservative nature. The overall structural similarity of these proteins provides an explanation for the antigenic cross-reactivity observed among diverse members of the HTLV retrovirus family by procedures that assay for the viral envelope glycoprotein, for example, membrane immunofluorescence.