The Combination of gQ1 and gQ2 in Human Herpesvirus 6A and 6B Regulates the Viral Tetramer Function for Their Receptor Recognition.

Human herpesvirus 6A (HHV-6A) and HHV-6B use different cellular receptors, human CD46 and CD134, respectively and have different cell tropisms although they have 90% similarity at the nucleotide level. An important feature that characterizes HHV-6A/6B is the glycoprotein H (gH)/gL/gQ1/gQ2 complex (a tetramer) that each virus has specifically on its envelope. Here, to determine which molecules in the tetramer contribute to the specificity for each receptor, we developed a cell-cell fusion assay system for HHV-6A and HHV-6B that uses the cells expressing CD46 or CD134. With this system, when we replaced the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion activity mediated by glycoproteins via CD46 was lower than that done with the original-type tetramer. When we replaced the gQ1 or the gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion mediated by glycoproteins via CD134 was not seen. In addition, we generated two types of C-terminal truncation mutants of HHV-6A gQ2 (AgQ2) to examine the interaction domains of HHV-6A gQ1 (AgQ1) and AgQ2. We found that amino acid residues 163 to 185 in AgQ2 are important for interaction of AgQ1 and AgQ2. Finally, to investigate whether HHV-6B gQ2 (BgQ2) can complement AgQ2, an HHV-6A genome harboring BgQ2 was constructed. The mutant could not produce an infectious virus, indicating that BgQ2 cannot work for the propagation of HHV-6A. These results suggest that gQ2 supports the tetramer's function, and the combination of gQ1 and gQ2 is critical for virus propagation.IMPORTANCE Glycoprotein Q2 (gQ2), an essential gene for virus propagation, forms a heterodimer with gQ1. The gQ1/gQ2 complex has a critical role in receptor recognition in the gH/gL/gQ1/gQ2 complex (a tetramer). We investigated whether gQ2 regulates the specific interaction between the HHV-6A or -6B tetramer and CD46 or CD134. We established a cell-cell fusion assay system for HHV-6A/6B and switched the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer. Although cell fusion was induced via CD46 when gQ1 or gQ2 was switched between HHV-6A and -6B, the activity was lower than that of the original combination. When gQ1 or gQ2 was switched in HHV-6A and -6B, no cell fusion was observed via CD134. HHV-6B gQ2 could not complement the function of HHV-6A's gQ2 in HHV-6A propagation, suggesting that the combination of gQ1 and gQ2 is critical to regulate the specificity of the tetramer's function for virus entry.

Comprehensive Screening of Mouse T-Cell Epitopes in Human Herpesvirus 6B Glycoprotein H/L/Q1/Q2 Tetramer Complex.

Human herpesvirus 6 (HHV-6) infects over 90% of people. The HHV-6 subtype, HHV-6B in particular, is often associated with exanthem subitum in early childhood. Exanthem subitum is usually self-limiting and good prognosis disease; however, some infants primarily infected with HHV-6B develop encephalitis/encephalopathy, and half of the patients developed encephalopathy reported to have neurological sequelae. Furthermore, after primary infection, HHV-6B remains in a latent state and sometimes reactivated in immunosuppressed patients, causing life-threatening severe encephalopathy. However, effective immunotherapies or vaccines for controlling HHV-6B infection and reactivation have not yet been established. Recently, we have found that the HHV-6B tetrameric glycoprotein (g) complex, gH/gL/gQ1/gQ2 is a promising vaccine candidate, and currently under preclinical development. To confirm our vaccine candidate protein complex induce detectable T-cell responses, in this study, we comprehensively screened CD4+ and CD8+ T-cell epitopes in the gH/gL/gQ1/gQ2 tetrameric complex protein in mice immunisation model. Both BALB/c and C57BL/6 mice were immunised with the tetrameric complex protein or plasmid DNA encoding gH, gL, gQ1, and gQ2, and then restimulated with 162 20-mer peptides covering the whole gH/gL/gQ1/gQ2 sequences; multiple CD4+ and CD8+ T-cell-stimulating peptides were identified in both BALB/c and C57BL/6 mice. Our study demonstrates that gH/gL/gQ1/gQ2 tetramer-targeted vaccination has potential to induce T-cell responses in two different strains of mice and supports the future development and application of T-cell-inducing vaccine and immunotherapies against HHV-6B.

Structural basis for the interaction of human herpesvirus 6B tetrameric glycoprotein complex with the cellular receptor, human CD134.

A unique glycoprotein is expressed on the virus envelope of human herpesvirus 6B (HHV-6B): the complex gH/gL/gQ1/gQ2 (hereafter referred to as the HHV-6B tetramer). This tetramer recognizes a host receptor expressed on activated T cells: human CD134 (hCD134). This interaction is essential for HHV-6B entry into the susceptible cells and is a determinant for HHV-6B cell tropism. The structural mechanisms underlying this unique interaction were unknown. Herein we solved the interactions between the HHV-6B tetramer and the receptor by using their neutralizing antibodies in molecular and structural analyses. A surface plasmon resonance analysis revealed fast dissociation/association between the tetramer and hCD134, although the affinity was high (KD = 18 nM) and comparable to those for the neutralizing antibodies (anti-gQ1: 17 nM, anti-gH: 2.7 nM). A competition assay demonstrated that the anti-gQ1 antibody competed with hCD134 in the HHV-6B tetramer binding whereas the anti-gH antibody did not, indicating the direct interaction of gQ1 and hCD134. A single-particle analysis by negative-staining electron microscopy revealed the tetramer's elongated shape with a gH/gL part and extra density corresponding to gQ1/gQ2. The anti-gQ1 antibody bound to the tip of the extra density, and anti-gH antibody bound to the putative gH/gL part. These results highlight the interaction of gQ1/gQ2 in the HHV-6B tetramer with hCD134, and they demonstrate common features among viral ligands of the betaherpesvirus subfamily from a macroscopic viewpoint.

Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B.

Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine.

Inhibitory effect of amenamevir on acute herpetic pain and postherpetic neuralgia in mice infected with herpes simplex virus-1.

BACKGROUND: Amenamevir (AMNV) is a helicase-primase inhibitor with antiviral activity against herpesviruses [herpes simplex viruses (HSV)-1 and -2, and varicella-zoster virus], which are associated with the development of acute herpetic pain (AHP) and postherpetic neuralgia. However, the inhibitory effects of helicase-primase inhibitors on AHP and postherpetic neuralgia remain incompletely understood. OBJECTIVE: In this study, we investigated the effects of AMNV on AHP and postherpetic pain (PHP) in HSV-1-infected mice accompanied by zosteriform-like skin lesions. METHODS: HSV-1 was percutaneously infected on the femoral region of mice. AMNV was orally administered twice a day for 5 days. Pain-related response in the hind paw was evaluated using a paintbrush. The infiltration of inflammatory cells in dorsal root ganglion (DRG) and spinal cord (SC) was evaluated by hematoxylin and eosin staining. The viral load in DRG and the expression of pain-related genes in SC were measured by real-time PCR. RESULTS: Pain response was begun to be observed from day 3 post-infection (pi) in HSV-1-infected mice. AMNV administered repeatedly from day 3 pi or day 4 pi, but not day 5 pi, showed an inhibitory effect on the development of AHP and the transition to PHP. Repeated AMNV administration inhibited inflammatory cell infiltration and increases in the viral load and the expression of pain-related genes (ATF-3, TNF-α, COX-2). CONCLUSION: These results demonstrate that AMNV potently suppresses the development of AHP and the transition to PHP as a consequence of decreased viral load in DRG and reduced expression of pain-related genes in SC.

Identification of CD4 and H-2Kd-restricted cytotoxic T lymphocyte epitopes on the human herpesvirus 6B glycoprotein Q1 protein.

The identification of Human herpesvirus 6B (HHV-6B) epitopes that are recognized by T-cells could contribute to the development of potential vaccines and immunotherapies. Here, we identified CD4+ and H-2Kd-restricted CD8+ T-cell epitopes on the glycoprotein Q1 of HHV-6B (BgQ1), which is a unique glycoprotein and essential for HHV-6B viral entry, by using in vivo electroporation with a plasmid DNA encoding BgQ1, overlapping peptides spanning the BgQ1 sequence, ELISPOT assay for quantification of gamma interferon (IFN-γ), and computer-based T-cell epitope prediction programs. The CD4+ and CD8+ T-cell epitopes identified in BALB/c mice in this study could be a good animal model system for use in the development of T-cell responses, inducing HHV-6B vaccines or immunotherapies.

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). OBJECTIVES: Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. STUDY DESIGN: Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4+ and CD8+ cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. RESULTS: HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4+ T cells, p = .02 in CD8+ T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4+ T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). CONCLUSIONS: This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT.

The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity.

Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity.IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents, suggesting that it might be a target of the immune system.

Comprehensive analysis of serum cytokines/chemokines in febrile children with primary human herpes virus-6B infection.

Cytokines and chemokines induced by primary human herpes virus (HHV)-6B infection may play a critical role in the clinical manifestations of infection. In this study, we analyzed 40 cytokines/chemokines in febrile children with primary HHV-6B infection. Blood samples from 233 febrile and 36 afebrile patients 0-3 years of age were used for this study. In febrile patients, primary HHV-6B infection was determined by detection of HHV-6B DNA without anti-HHV-6 immunoglobulin G in the blood (HHV-6B group). Infection by other pathogens was assumed when HHV-6B DNA was not detected in the blood (non-HHV-6B group). Of the 233 febrile patients, 30 patients (13%) were diagnosed with primary HHV-6B infection. To analyze serum cytokines/chemokines, patients were randomly chosen from the HHV-6B (n = 25) and non-HHV-6B groups (n = 8). Sera from 25 afebrile patients were used as a control. When comparing the levels of 40 cytokines/chemokines between the HHV-6B and control groups, we found that four chemokines (chemokine [C-X-C motif] ligand [CXCL] 11, CXCL10, CXCL16, and chemokine [C-C motif] ligand [CCL] 2) were significantly upregulated in the HHV-6B group compared with those in the control. Of these, only CXCL11 levels were significantly higher in the HHV-6B group than in the non-HHV-6B group. Because the induction of CCL2 was already reported in an early study, we found, for the first time, the induction of three new chemokines, i.e., CXCL11, CXCL10, and CXCL16 in patients with primary HHV-6B infection. Importantly, we demonstrated that serum CXCL11 levels increased specifically in patients with HHV-6B infection.

Crystal Structure of Human Herpesvirus 6B Tegument Protein U14.

The tegument protein U14 of human herpesvirus 6B (HHV-6B) constitutes the viral virion structure and is essential for viral growth. To define the characteristics and functions of U14, we determined the crystal structure of the N-terminal domain of HHV-6B U14 (U14-NTD) at 1.85 Å resolution. U14-NTD forms an elongated helix-rich fold with a protruding ß hairpin. U14-NTD exists as a dimer exhibiting broad electrostatic interactions and a network of hydrogen bonds. This is first report of the crystal structure and dimerization of HHV-6B U14. The surface of the U14-NTD dimer reveals multiple clusters of negatively- and positively-charged residues that coincide with potential functional sites of U14. Three successive residues, L424, E425 and V426, which relate to viral growth, reside on the ß hairpin close to the dimer's two-fold axis. The hydrophobic side-chains of L424 and V426 that constitute a part of a hydrophobic patch are solvent-exposed, indicating the possibility that the ß hairpin region is a key functional site of HHV-6 U14. Structure-based sequence comparison suggests that U14-NTD corresponds to the core fold conserved among U14 homologs, human herpesvirus 7 U14, and human cytomegalovirus UL25 and UL35, although dimerization appears to be a specific feature of the U14 group.

Human herpesvirus 6 U11 protein is critical for virus infection.

All herpesviruses contain a tegument layer comprising a protein matrix; these proteins play key roles during viral assembly and egress. Here, liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) of proteins from human herpesvirus 6 (HHV-6)-infected cells revealed a possible association between two major tegument proteins, U14 and U11. This association was verified by immunoprecipitation experiments. Moreover, U11 protein was expressed during the late phase of infection and incorporated into virions. Finally, in contrast to its revertant, a U11 deletion mutant could not be reconstituted. Taken together, these results suggest that HHV-6 U11 is an essential gene for virus growth and propagation.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection.

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation.

Human Herpesvirus 6A U14 Is Important for Virus Maturation.

Human herpesvirus 6A (HHV-6A) U14 is a virion protein with little known function in virus propagation. Here, we elucidated its function by constructing and analyzing U14-mutated viruses. We found that U14 is essential for HHV-6A propagation. We then constructed a mutant virus harboring dysfunctional U14. This virus showed severely reduced growth and retarded maturation. Taken together, these data indicate that U14 plays an important role during HHV-6A maturation.

Human Herpesvirus-6 U14 Induces Cell-Cycle Arrest in G2/M Phase by Associating with a Cellular Protein, EDD.

The human herpesvirus-6 (HHV-6) infection induces cell-cycle arrest. In this study, we found that the HHV-6-encoded U14 protein induced cell-cycle arrest at G2/M phase via an association with the cellular protein EDD, a mediator of DNA-damage signal transduction. In the early phase of HHV-6 infection, U14 colocalized with EDD dots in the nucleus, and similar colocalization was also observed in cells transfected with a U14 expression vector. When the carboxyl-terminal region of U14 was deleted, no association of U14 and EDD was observed, and the percentage of cells in G2/M decreased relative to that in cells expressing wild-type U14, indicating that the C-terminal region of U14 and the U14-EDD association are critical for the cell-cycle arrest induced by U14. These results indicate that U14 is a G2/M checkpoint regulator encoded by HHV-6.

The human herpesvirus 6 U21-U24 gene cluster is dispensable for virus growth.

Human herpesvirus 6 (HHV-6) is a T-lymphotrophic virus belongs to the genus Roseolovirus within the beta herpesvirus subfamily. The U20-U24 gene cluster is unique to Roseoloviruses; however, both their function and whether they are essential for virus growth is unknown. Recently, bacterial artificial chromosome (BAC) techniques have been used to investigate HHV-6A. This study describes generation of a virus genome lacking U21-U24 (HHV-6ABACΔU21-24) and shows that infectious virus particles can be reconstituted from this BAC DNA. Our data indicate that the HHV-6 U21-U24 gene cluster is dispensable for virus propagation.

Human herpesvirus 6 gM/gN complex interacts with v-SNARE in infected cells.

Human herpesvirus 6 (HHV-6) glycoprotein M (gM) is an envelope glycoprotein that associates with glycoprotein N (gN), forming the gM/gN protein complex, in a similar manner to the other herpesviruses. Liquid chromatography-MS/MS analysis showed that the HHV-6 gM/gN complex interacts with the v-SNARE protein, vesicle-associated membrane protein 3 (VAMP3). VAMP3 colocalized with the gM/gN complex at the trans-Golgi network and other compartments, possibly the late endosome in HHV-6-infected cells, and its expression gradually increased during the late phase of virus infection. Finally, VAMP3 was incorporated into mature virions and may be transported with the gM/gN complex.

Characterization of the human herpesvirus 6A U23 gene.

Human herpesvirus 6 (HHV-6), which replicates abundantly in T cells, belongs to the Roseolovirus genus within the betaherpesvirus subfamily. Members of the Roseolovirus genus encode seven unique genes, U20, U21, U23, U24, U24A, U26, and U100. The present study focused on one of these, U23, by analyzing the characteristics of its gene product in HHV-6A-infected cells. The results indicated that the U23 protein was expressed at the late phase of infection as a glycoprotein, but was not incorporated into virions, and mostly stayed within the trans Golgi network (TGN) in HHV-6A-infected cells. Furthermore, analysis using a U23-defective mutant virus showed that the gene is nonessential for viral replication in vitro.

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

Human herpesvirus-6B (HHV-6B) is a T lymphotropic ß-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.

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 M is essential for virus growth and requires glycoprotein N for its maturation.

Human herpesvirus 6 (HHV-6) is a T-lymphotropic virus belonging to the betaherpesvirus family. Several HHV-6-encoded glycoproteins are required for cell entry and virion maturation. Glycoprotein M (gM) is conserved among all herpesviruses, and therefore thought to have important functions; however, the HHV-6 g has not been characterized. Here, we examined the expression of HHV-6 g, and examined its function in viral replication, using a mutant and revertant gM. HHV-6 g was expressed on virions as a glycoprotein modified with complex N-linked oligosaccharides. As in other herpesviruses, HHV-6 g formed a complex with glycoprotein N (gN), and was transported from the endoplasmic reticulum to the trans-Golgi network only when part of this complex. Finally, a gM mutant virus in which the gM start codon was destroyed was not reconstituted, although its revertant was, indicating that HHV-6 g is essential for virus production, unlike the gM of alphaherpesviruses.