Recent topics related to human herpesvirus 6 cell tropism

Human herpesvirus 6 (HHV-6) is a T lymphotropic herpes virus that is categorized into two variants, A (HHV-6A) and B (HHV-6B), on the basis of distinct genetic, immunological and biological characteristics. HHV-6 uses human CD46 as a cellular receptor. Without viral replication, HHV-6A induces cell–cell fusion between cells expressing human CD46. Some HHV-6B strains can also induce CD46-mediated cell–cell fusion. A multiple glycoprotein complex composed of glycoprotein (g) H-gL complexed with gQ1 and gQ2 has been identified, and found to be a viral ligand for the human CD46 receptor. Moreover, a novel complex consisting of gH/gL/gO, which does not associate with CD46, has also been identified. The evidence suggests that an additional receptor for HHV-6B or both variants may play a role in determining the cell tropism of this virus. Finally, cholesterol in the HHV-6 envelope and plasma membrane of the host cells plays an important role in HHV-6 entry, although how this function relates to cell–envelope fusion remains to be elucidated.     

Identification of the Human Herpesvirus 6A gQ1 Domain Essential for Its Functional Conformation

Human herpesvirus 6 is a T lymphotropic herpesvirus, long classified into variants A and B (HHV-6A and HHV-6B) based on differences in sequence and pathogenicity. Recently, however, HHV-6A and HHV-6B were reclassified as different species. Here, we isolated a neutralizing monoclonal antibody (Mab) named AgQ 1-1 that was specific for HHV-6A glycoprotein Q1 (AgQ1), and we showed that amino acid residues 494 to 497 of AgQ1 were critical for its recognition by this Mab. This region was also essential for AgQ1''s complex formation with gH, gL, and gQ2, which might be important for viral binding to the cellular receptor, CD46. In addition, amino acid residues 494 to 497 are essential for viral replication. Interestingly, this sequence corresponds to the domain on HHV-6B gQ1 that is critical for recognition by an HHV-6B-specific neutralizing Mab. Within this domain, only Q at position 496 of HHV-6A is distinct from the HHV-6B sequence; however, the mutant AgQ1(Q496E) was still clearly recognized by the Mab AgQ 1-1. Surprisingly, replacement of the adjacent amino acid, in mutant AgQ1(C495A), resulted in poor recognition by Mab AgQ 1-1, and AgQ1(C495A) could not form the gH/gL/gQ1/gQ2 complex. Furthermore, the binding ability of mutant AgQ1(L494A) with CD46 decreased, although it could form the gH/gL/gQ1/gQ2 complex and it showed clear reactivity to Mab AgQ 1-1. These data indicated that amino acid residues 494 to 497 of AgQ1 were critical for the recognition by Mab AgQ 1-1 and essential for AgQ1''s functional conformation.     

Human herpesvirus 6 glycoprotein complex formation is required for folding and trafficking of the gH/gL/gQ1/gQ2 complex and its cellular receptor binding

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. A glycoprotein (g) complex that is unique to HHV-6, gH/gL/gQ1/gQ2, is a viral ligand for its cellular receptor, human CD46. However, whether complex formation or one component of the complex is required for CD46 binding and how the complex is transported in cells are open questions. Furthermore, in HHV-6-infected cells the gQ1 protein modified with N-linked glycans is expressed in two forms with different molecular masses: an 80-kDa form (gQ1-80K) and a 74-kDa form (gQ1-74K). Only gQ1-80K, but not gQ1-74K, forms the complex with gQ2, gH, and gL, and this four-component complex is incorporated into mature virions. Here, we characterized the molecular context leading to the maturation of gQ1 by expressing combinations of the individual gH/gL/gQ1/gQ2 components in 293T cells. Surprisingly, only when all four molecules were expressed was a substantial amount of gQ1-80K detected, indicating that all three of the other molecules (gQ2, gH, and gL) were necessary and sufficient for gQ1 maturation. We also found that only the tetrameric complex, and not its subsets, binds to CD46. Finally, a gQ2-null virus constructed in the BAC (bacterial artificial chromosome) system could not be reconstituted, indicating that gQ2 is essential for virus growth. These results show that gH, gL, gQ1, and gQ2 are all essential for the trafficking and proper folding of the gH/gL/gQ1/gQ2 complex and, thus, for HHV-6 infection.     

Herpesvirus 6 Glycoproteins B (gB), gH,gL, and gQ Are Necessary and Sufficient for Cell-to-Cell Fusion

The human herpesvirus 6 (HHV-6) envelope glycoprotein gH/gL/gQ1/gQ2 complex associates with host cell CD46 as its cellular receptor. Although gB has been suggested to be involved in HHV-6 infection, its function in membrane fusion has remained unclear. Here, we have developed an HHV-6A (strain GS)and HHV-6B (strain Z29) virus-free cell-to-cell fusion assay and demonstrate that gB and the gH/gL/gQ1/gQ2 complex are the minimum components required for membrane fusion by HHV-6.     

Discovery of a second form of tripartite complex containing gH-gL of human herpesvirus 6 and observations on CD46

The human herpesvirus 6 (HHV-6) glycoprotein H (gH)-glycoprotein L (gL) complex associates with glycoprotein Q (gQ) (Y. Mori, P. Akkapaiboon, X. Yang, and K. Yamanishi, J. Virol. 77:2452-2458, 2003), and the gH-gL-gQ complex interacts with human CD46 (Y. Mori, X. Yang, P. Akkapaiboon, T. Okuno, and K. Yamanishi, J. Virol. 77:4992-4999, 2003). Here, we show that the HHV-6 U47 gene, which is a positional homolog of the human cytomegalovirus glycoprotein O (gO) gene, encodes a third component of the HHV-6 gH-gL-containing envelope complex. A monoclonal antibody (MAb) against the amino terminus of HHV-6 gO reacted in immunoblots with protein species migrating at 120 to 130 kDa and 74 to 80 kDa in lysates of HHV-6-infected cells and with a 74- to 80-kDa protein species in purified virions. The 80-kDa form of gO was coimmunoprecipitated with an anti-gH MAb, but an anti-gQ MAb, which coimmunoprecipitated gH, did not coprecipitate gO. Furthermore, the gH-gL-gO complex did not bind to human CD46, indicating that the complex was not a ligand for CD46. These findings suggested that the viral envelope contains at least two kinds of tripartite complexes, gH-gL-gQ and gH-gL-gO, and that the gH-gL-gO complex may play a role different from that of gH-gL-gQ during viral infection. This is the first report of two kinds of gH-gL complexes on the viral envelope in a member of the herpesvirus family.     

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.     

Complementation of the function of glycoprotein H of human herpesvirus 6 variant A by glycoprotein H of variant B in the virus life cycle

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle.     

Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site

We have investigated whether nonneutralizing monoclonal antibodies (MAbs) to the gp120 subunit of the envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 (HIV-1) can interfere with HIV-1 neutralization by another anti-gp120 MAb. We used neutralizing (b12) and nonneutralizing (205-42-15, 204-43-1, 205-46-9) MAbs to the epitope cluster overlapping the CD4-binding site (CD4BS) on gp120. All the MAbs, neutralizing or otherwise, cross-competed for binding to monomeric gp120, indicating the close topological proximity of their epitopes. However, the nonneutralizing CD4BS MAbs did not interfere with the neutralization activity of MAb b12. In contrast, in a binding assay using oligomeric Env expressed on the surface of Env-transfected cells, the nonneutralizing MAbs did partially compete with b12 for Env binding. The surface of Env-transfected cells contains two categories of binding site for CD4BS MAbs. One type of site is recognized by both b12 and nonneutralizing CD4BS MAbs; the other is recognized by only b12. Binding assays for Env-gp120 interactions based on the use of monomeric gp120 or Env-transfected cells do not predict the outcome of HIV-1 neutralization assays, and they should therefore be used only with caution when gauging the properties of anti-Env MAbs.     

脂筏在人类疱疹病毒6型装配中的作用

为了探讨脂筏在人类疱疹病毒6型(HHV-6)装配中的作用,用HHV-6 GS株感染HSB2细胞,用非离子去污剂Triton X-100提取脂筏成分,利用Western blot分析HHV-6包膜糖蛋白与脂筏的相关性.并用免疫荧光双标记的方法,从分子共定位的角度研究HHV-6糖蛋白B(gB)与GPI(glycosyl-phosphatidyl inosital)锚固蛋白CD59分子以及神经节苷脂GMI(monosialotetrahexosyl ganglioside)分子之间的表达与分布关系.结果发现HHV-6包膜糖蛋白B、H、L、Q1和Q2(gB、gH、gL、gQ1和gQ2)分布在脂筏部位.激光共聚焦显微镜可观察到CD59分子及GM1均与HHV-6包膜糖蛋白B有着相同的分布,即脂筏提供HHV-6装配的平台.关于脂筏在人类疱疹病毒6型装配中的作用,这是第一次报道.     

Human herpesvirus 6 variant A glycoprotein H-glycoprotein L-glycoprotein Q complex associates with human CD46

Human CD46 is a cellular receptor for human herpesvirus 6 (HHV-6). Virus entry into host cells requires a glycoprotein H (gH)-glycoprotein L (gL) complex. We show that the CD46 ectodomain blocked HHV-6 infection and bound a complex of gH-gL and the 80-kDa U100 gene product, designated glycoprotein Q, indicating that the complex is a viral ligand for CD46.     

Human herpesvirus‐6A gQ1 and gQ2 are critical for human CD46 usage

Based on genetic and antigenic differences and on their cell tropism, human herpes virus‐6 (HHV‐6) has been classified into two variants, HHV‐6A and HHV‐6B. Recently, these variants were re‐classified as two different species. The HHV‐6A glycoprotein complex, gH/gL/gQ1/gQ2 binds to its cellular receptor, CD46; however, the corresponding complex in HHV‐6B rarely binds to CD46. To determine which viral molecules in the glycoprotein complex determine HHV‐6A‐CD46 binding, each molecule of the HHV‐6A complex (i.e., gH, gL, gQ1, or gQ2) was replaced with the corresponding HHV‐6B molecule, and the ability of the replaced protein to be incorporated into the complex and the ability of the complex to bind CD46 were examined. It was found that when all four glycoproteins were expressed, they were able to form a tetrameric complex. However, a complex formed by HHV‐6A gH/gL/gQ1/gQ2 complexes replaced with HHV‐6B gQ1 or gQ2 scarcely bind CD46, whereas HHV‐6A complexes in which gH or gL was replaced with the HHV‐6B molecules did bind it. These results indicate that HHV‐6A gQ1 and gQ2 play an important role in CD46 binding.     

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.     

Human herpesvirus 6 variant A but not variant B induces fusion from without in a variety of human cells through a human herpesvirus 6 entry receptor,CD46

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.     

Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46

Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an approximately 110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).     

Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus.

The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells.     

Identification and characterization of an immunogenic hybrid epitope formed by both HIV gp120 and human CD4 proteins

Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection.     

Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles

The selection of human monoclonal antibodies (MAbs) specific for human immunodeficiency virus (HIV) type 1 by binding assays may fail to identify Abs to quaternary epitopes on the intact virions. The HIV neutralization assay was used for the selection of human MAb 2909, which potently neutralizes SF162 and recognizes an epitope on the virus surface but not on soluble proteins. Three regions of gp120, the V2 and V3 loops and the CD4 binding domain, contribute to the epitope recognized by MAb 2909. The existence of such a unique MAb, which defines a complex epitope formed by a quaternary structure, suggests that there may be other new neutralizing HIV epitopes to target with vaccines.