Huamin Tang

Human herpesvirus-6 induces MVB formation, and virus egress occurs by an exosomal release pathway.

The final envelopment of most herpesviruses occurs at Golgi or post‐Golgi compartments, such as the trans Golgi network (TGN); however, the final envelopment site of human herpesvirus 6 (HHV‐6) is uncertain. In this study, we found novel pathways for HHV‐6 assembly and release from T cells that differed, in part, from those of alphaherpesviruses. Electron microscopy showed that late in infection, HHV‐6‐infected cells were larger than uninfected cells and contained many newly formed multivesicular body (MVB)‐like compartments that included small vesicles. These MVBs surrounded the Golgi apparatus. Mature virions were found in the MVBs and MVB fusion with plasma membrane, and the release of mature virions together with small vesicles was observed at the cell surface. Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB/late endosome marker, and HHV‐6 envelope glycoproteins. The viral glycoproteins also localized to internal vesicles in the MVBs and to secreted vesicles (exosomes). Furthermore, we found virus budding at TGN‐associated membranes, which expressed CD63, adaptor protein (AP‐1) and TGN46, and CD63 incorporation into virions. Our findings suggest that mature HHV‐6 virions are released together with internal vesicles through MVBs by the cellular exosomal pathway. This scenario has significant implications for understanding HHV‐6’s maturation pathway.

CD134 is a cellular receptor specific for human herpesvirus-6B entry

Human herpesvirus-6B (HHV-6B) is a T lymphotropic β-herpesvirus that is clearly distinct from human herpesvirus-6A (HHV-6A) according to molecular biological features. The International Committee on Taxonomy of Viruses recently classified HHV-6B as a separate species. The primary HHV-6B infection causes exanthem subitum and is sometimes associated with severe encephalopathy. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection. HHV-6B reactivation causes encephalitis in immunosuppressed patients. The cellular receptor for HHV-6A entry was identified as human CD46, but the receptor for HHV-6B has not been clear. Here we found that CD134, a member of the TNF receptor superfamily, functions as a specific entry receptor for HHV-6B. A T-cell line that is normally nonpermissive for HHV-6B infection became highly susceptible to infection when CD134 was overexpressed. CD134 was down-regulated in HHV-6B–infected T cells. Soluble CD134 interacted with the HHV-6B glycoprotein complex that serves as a viral ligand for cellular receptor, which inhibited HHV-6B but not HHV-6A infection in target cells. The identification of CD134 as an HHV-6B specific entry receptor provides important insight into understanding HHV-6B entry and its pathogenesis.

Human herpesvirus 6 encoded glycoprotein Q1 gene is essential for virus growth

Human herpesvirus 6 (HHV-6) glycoprotein Q1 (gQ1), a unique gene in HHV-6, forms a complex with glycoproteinH (gH) and gL, which is the viral ligand for its cellular receptor, CD46. However, whether gQ1 is essential for virus growth is unknown, because a system is lacking for making gene knockouts for HHV-6. Recently, bacterial artificial chromosome (BAC) and E. coli mutagenesis techniques have been applied to herpesvirus investigation. Here we successfully inserted the HHV-6A genome into a BAC, and obtained reconstituted infectious virus from the HHV-6A-containing BAC DNA. Using this system, we generated a gQ1 mutant virus genome, which failed to yield reconstituted infectious virus, whereas its revertant virus could be produced, indicating that the HHV-6 gQ1 gene is essential for virus growth. Therefore, we successfully applied BAC and E. coli mutagenesis techniques to the study of HHV-6, and discovered that HHV-6 gQ1 is an essential gene for virus growth.

Human herpesvirus-6 infection induces the reorganization of membrane microdomains in target cells, which are required for virus entry.

Cell-membrane raft microdomains are important for successful infection by several viruses. However, their role in the cell-entry process of human herpesvirus-6 (HHV-6) is unknown. Here we tested whether HHV-6 requires cell-membrane rafts for its entry. When cell-membrane rafts were disrupted by cholesterol depletion, target-cell entry by HHV-6 was inhibited, although the virus bound normally to the cells. HHV-6 infectivity was partially rescued by adding exogenous cholesterol. Interestingly, the HHV-6 cellular receptor, CD46, was found in the rafts after virus attachment, but not in the rafts of uninfected cells, indicating that HHV-6 infection induces the re-location of its receptor into the rafts. Furthermore, glycoprotein Q1, part of a viral glycoprotein complex that binds CD46, was also associated with rafts immediately after infection. These data suggest that cellular-membrane lipid rafts are important in viral entry and that HHV-6 may enter the target cells via the rafts.

Human Herpesvirus 6 Glycoprotein Complex Formation Is Required for Folding and Trafficking of the gH/gL/gQ1/gQ2 Complex and Its Cellular Receptor Binding

ABSTRACT 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.

Complementation of the Function of Glycoprotein H of Human Herpesvirus 6 Variant A by Glycoprotein H of Variant B in the Virus Life Cycle

ABSTRACT 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.

Analysis of a neutralizing antibody for human herpesvirus-6B reveals a role for glycoprotein Q1 in viral entry

ABSTRACT Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.

Human herpesvirus-6 entry into host cells

Human herpesvirus-6 (HHV-6) belongs to the herpesvirus family and is categorized into variant A and B (HHV-6A and HHV-6B). Primary HHV-6 infection in children and its related diseases are almost exclusively caused by HHV-6B and no disease caused by HHV-6A has been identified. The cellular receptor of HHV-6 has been shown to be a human CD46, and its viral ligand is an envelope glycoprotein complex, gH/gL/gQ1/gQ2 in HHV-6A. Furthermore, both cellular and viral lipid rafts play an important role in the HHV-6 entry process, suggesting that HHV-6 may enter its target cells through a lipid raft-associated mechanism.

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.

Human herpesvirus 6 envelope components enriched in lipid rafts: evidence for virion-associated lipid rafts

In general, enveloped viruses are highly dependent on their lipid envelope for entry into host cells. Here, we demonstrated that during the course of virus maturation, a significant proportion of human herpesvirus 6 (HHV-6) envelope proteins were selectively concentrated in the detergent-resistant glycosphingolipid- and cholesterol-rich membranes (rafts) in HHV-6-infected cells. In addition, the ganglioside GM1, which is known to partition preferentially into lipid rafts, was detected in purified virions, along with viral envelope glycoproteins, gH, gL, gB, gQ1, gQ2 and gO indicating that at least one raft component was included in the viral particle during the assembly process.