B5R Gene Research Articles

Microarray assay for evaluation of the genetic stability of modified vaccinia virus Ankara B5R gene

Adverse events associated with the use of live smallpox vaccines have led to the development of a new generation of attenuated smallpox vaccines that are prepared in cultured cells as alternatives. The inability to conduct direct clinical evaluation of their efficacy in humans demands that licensure be based on animal studies and exhaustive evaluation of their in vitro properties. One of the most important characteristics of live viral vaccines is their genetic stability, including reversion of the vaccine strain to more virulent forms, recombination with other viral sequences to produce potentially pathogenic viruses, and genetic drift that can result in decrease of immunogenicity and efficacy. To study genetic stability of an immunoessential vaccinia virus gene in a new generation smallpox vaccine, an advanced oligonucleotide microchip was developed and used to assay for mutations that could emerge in B5R gene, a vaccinia virus gene encoding for a protein that contains very important neutralizing epitopes. This microarray contained overlapping oligonucleotides covering the B5R gene of modified vaccinia virus Ankara (MVA), a well-studied candidate smallpox vaccine. The microarray assay was shown to be able to detect even a single point mutation, and to differentiate between vaccinia strains. At the same time, it could detect newly emerged mutations in clones of vaccinia strains. In the work described here, it was shown that MVA B5R gene was stable after 34 passages in Vero and MRC-5 cells that were proposed for use as cell substrates for vaccine manufacture. Potentially, the proposed method could be used as an identity test and could be extended for the entire viral genome and used to monitor consistency of vaccine production.

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-gamma receptor homolog (viroceptor). To facilitate the monitoring of virus properties, first we constructed a Lister vaccine strain derivative VV-RG expressing optical reporters. Further, we constructed a VV-RG derivative, VV-RG8, which lacks the IFN-gammaR viroceptor (B8R gene product). Replication, immunological and pathogenic properties of the constructed strains were compared. Viruses did not show significant differences in humoral and cellular immune responses of immune-competent mice. Replication of constructed viruses was efficient both in vitro and in vivo, but showed marked difference in kinetics of propagation. In cultured CV-1 epithelial cells, the VV-RG8 strain retained the propagation potential of the parental virus, while, in the C6 glial cells, significant delay was observed in the kinetics of the VV-RG8 replication cycle compared to VV-RG. The pathogenesis of the viruses was tested by survival assay and biodistribution in nude mice. High dose inoculation of nude mice with VV-RG8 caused less pronounced virus dissemination, improved weight gain, and increased survival rate, as compared with the VV-RG strain. The replication-competent virus VV-RG8 carrying a mutation at the B8R gene is less pathogenic for mice than the parental vaccine virus. We anticipate that step-wise inactivation of VV vaccine genes involved in evasion of host immune response may provide an alternative approach for generation of hyper-attenuated replication-competent vaccines.

Role of Genes That Modulate Host Immune Responses in the Immunogenicity and Pathogenicity of Vaccinia Virus

Poxvirus vaccine vectors, although capable of eliciting potent immune responses, pose serious health risks in immunosuppressed individuals. We therefore constructed five novel recombinant vaccinia virus vectors which contained overlapping deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R, and B19R immunomodulatory gene products and assessed them for both immunogenicity and pathogenicity. All five of these novel vectors elicited both cellular and humoral immunity to the inserted HIV-BH10 env comparable to that induced by the parental Wyeth strain vaccinia virus. However, deletion of these immunomodulatory genes did not increase the immunogenicity of these vectors compared with the parental vaccinia virus. Furthermore, four of these vectors were slightly less virulent and one was slightly more virulent than the Wyeth strain virus in neonatal mice. Attenuated poxviruses have potential use as safer alternatives to current replication-competent vaccinia virus. Improved vaccinia virus vectors can be generated by deleting additional genes to achieve a more significant viral attenuation.

Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R

Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8.

A highly attenuated LC16m8 (m8) smallpox vaccine has been licensed in Japan because of its extremely low neurovirulence profile, which is comparable to that of replication incompetent strains of vaccinia virus. From 1973 to 1975, m8 was administrated to >100,000 infants where it induced levels of immunity similar to that of the originating Lister strain, without any serious side effects. Recently, we observed that m8 reverts spontaneously to large plaque forming clones that possess virulence equivalent to that of LC16mO, a parental virus strain of m8. Here, we report that the B5R gene is responsible for the reversion, and that we could construct a more genetically stable virus by deleting B5R from m8. The protective immunogenicity of the vaccine candidate proved to be equivalent to that of the U.S.-licensed product Dryvax, and much superior to modified vaccinia Ankara in a mouse model. Furthermore, the vaccine strain never elicited any symptoms in severe combined immunodeficiency disease mice, even at a dose 1,000-fold greater than that used in the immune protection experiments, which is in contrast to the lethal pathogenicity induced by Dryvax inoculation of severe combined immunodeficiency disease mice. Our results suggest that this vaccine strain is a good candidate as a suitable smallpox vaccine and a vector virus, and that B5R is not essential for protective immunity against smallpox.

Heterogeneity of Molecular Biology of Methemoglobinemia; Study of Eight Consecutive Patients.

Methemoglobinemia is a rare congenital or acquired disorder characterized by increased levels of oxidized hemoglobin in which the ferrous iron (Fe++) is in the ferric (Fe+++) state.Congenital methemoglobinemia can be due to mutations of a) NADH Cytochrome b5 reductase (b5R), either type I - deficiency restricted to RBCs; and causing cyanosis, or type II - deficiency in all cells;(fatal in childhood), b) globin genes or c) cytochrome b5. Eight consecutive methemoglobinemia patients had spectrometric methemoglobin analyses, b5R activity assay, and analysis of b5R mRNA and genomic DNA. Four patients had mutations of b5R gene; two were novel (see Table). Two had type I and 2 had type II b5R deficiency. One was a compound heterozygote and three were homozygotes; 2 were intronic and 3 were exonic mutations. The molecular consequences of both intronic mutations were characterized; we report a novel mechanism of markedly reduced b5R transcript causing clinical type II deficiency. We also show that in one patient with type II deficiency the diagnosis would be missed by measuring the erythrocyte b5R activity alone.Three patients had acquired toxic methemoglobinemia; all had normal b5R activity and no mutations involving the b5R gene. One patient had congenital methemoglobinemia caused by a beta globin mutation (Hb MHyde Park). We conclude that: a) congenital methemoglobinemia due to mutations of b5R gene is at least as common as acquired methemoglobinemia; b) contrary to previous reports, b5R heterozygosity is not a common predisposition for acquired methemoglobinemia; c) congenital methemoglobinemias due to globin mutations are rare; and d) screening by enzyme assay is not a reliable predictor of b5R deficiency.Summary of results. The mutations marked by * are novel.Pt#DiagnosisMethemoglobinCell type and B5R activityB5R gene mutationConsequences of mutation and remarks1Type I RCM12%RBC-20%1.22236 G →A in exon 6 and 2.22249 G →A* (IVS 6+1)1.178 Alanine → Threonine and 2. Aberrant mRNA processing with 3 different transcripts: a) in-frame skipping of exon 6 b) all exons +46 bp of intron 6 c) skipped exon 6 with 179 bp of intron 5. No normal transcript from this chromosome. Normal expression by Real time qPCR2Type I RCMNARBC-25%PLT, GNC and MNC - 100%29951 C →T*in exon 9258 Arginine → Tryptophan3Type II RCM30%RBC - 50%and EBV - 25%22526 T →C in exon 7203 Cysteine → Arginine4Type II RCM20%RBC - 2% and EBV - 25%22163 A →C (IVS 5–2)Aberrant mRNA processing with 4 different transcripts: a)in-frame skipping of exon 6, b) normal transcript c) 217 bp of intron 6 included d) entire intron 6 (463 bp) included. Reduced expression by Real time qPCR with only 7% of normal mRNA in the patient and 28% of normal mRNA in the heterozygote mother.5Infant Toxic35%RBC - 60%NoneOne week of diarrheal illness. Needed several administrations of methylene blue6Toxic12%RBC - 100%NoneLidocaine exposure7Toxic44%RBC - 100%NoneUnidentified toxin or infection8Hb.M- Hyde ParkNARBC - 100%Beta globin gene @ codon 92 C → TBeta globin 92 Histidine →TyrosineRCM - recessive congenital methemoglobinemia, EBV - EBV transformed lymphocytes, PLT - platelets, GNC - granulocytes, MNC - mononuclear cells, B5R gene mutations numbered as in NCBI accession AY341030

Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates

Two major infectious forms of vaccinia virus (VACV) have been described: the intracellular mature virion (IMV), and the extracellular enveloped virion (EEV). Due to their stability in the environment, IMVs play a predominant role in host-to-host transmission, whereas EEVs play an important role in dissemination within the host. In a previous report, we demonstrated that mice vaccinated with VACV L1R (IMV immunogen) and A33R (EEV immunogen) were protected from a lethal poxvirus challenge. Vaccination with a combination of both genes conferred greater protection than either gene alone, suggesting that an immune response against both IMV and EEV is advantageous. Here, we report that in mice individually administered DNA vaccines with two different VACV immunogens, A27L (IMV immunogen) or B5R (EEV immunogen), failed to significantly protect; however, vaccination with a combination of both genes conferred a high level of protection. Mice were completely protected when vaccinated with a combination of four VACV genes (A27L + A33R + L1R + B5R). Rhesus macaques vaccinated with this four-gene-combination developed appropriate antibody responses to each protein. Antibody responses elicited by this vaccine cross-reacted with monkeypox virus orthologous proteins. These data indicate that a gene-based vaccine comprised of the VACV A27L + A33R + L1R + B5R genes may be a useful candidate to protect against other orthopoxviruses, including those that cause monkeypox and smallpox.

A study of the vaccinia virus interferon-gamma receptor and its contribution to virus virulence.

Vaccinia virus (VV) strain Western Reserve gene B8R encodes a 43 kDa glycoprotein that is secreted from infected cells early in infection as a homodimer. This protein has amino acid similarity with the extracellular domain of cellular IFN-gamma receptor (IFN-gammaR) and binds and inhibits IFN-gamma from a wide range of species. Here we demonstrate that the B8R protein also inhibits equine IFN-gamma. The 5' end of the B8R mRNA has been mapped by primer extension analysis and the contribution of IFN-gammaRs to VV virulence was studied by the construction of a deletion mutant lacking the B8R gene (vDeltaB8R) and a revertant virus (vB8R-R) in which the B8R gene was re-inserted into the deletion mutant. A recombinant virus that expressed a soluble form of the mouse IFN-gammaR was also constructed and studied. The virulence of these viruses was tested in rodent models of infection. In mice, the loss of the VV IFN-gammaR did not affect virulence compared with WT and revertant viruses, consistent with the low affinity of the VV IFN-gammaR for mouse IFN-gamma. However, expression of the mouse soluble IFN-gammaR increased virus virulence slightly. In rabbit skin, loss of the VV IFN-gammaR produced lesions with histological differences compared with WT and revertant viruses. Lastly, the affinity constants of the VV IFN-gammaR for human and mouse IFN-gamma were determined by surface plasmon resonance.

A mutational analysis of the vaccinia virus B5R protein.

A mutational analysis of the vaccinia virus (VV) B5R protein is presented. This protein is related to the regulators of complement activation (RCA) superfamily, has four short consensus repeats (SCRs) that are typical of this superfamily and is present on extracellular enveloped virus (EEV) particles. Here we have constructed VV mutants in which the cytoplasmic tail (CT) of the B5R protein is progressively truncated, and domains of the B5R protein [the SCR (short consensus repeat) domains, the transmembrane anchor region or the CT] are substituted by corresponding domains from the VV haemagglutinin (HA), another EEV protein. Analysis of these mutant viruses showed that loss of the B5R CT did not affect the formation of intracellular enveloped virus (IEV), actin tails, EEV or virus plaque size. However, if the SCR domains of the B5R protein were replaced by the corresponding region of the HA, the virus plaque size was diminished, the formation of actin tails was decreased severely and the titre of infectious EEV released from cells was reduced approximately 25-fold compared to wild-type virus and 5-fold compared to a virus lacking the entire B5R gene. Thus the linkage of HA to the B5R transmembrane and CT is deleterious for the formation and release of EEV and for cell-to-cell virus spread. In contrast, deletion or substitution of the B5R CT did not affect virus replication, although the amount of cell surface B5R was reduced compared to control.

Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans.

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S' and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS'-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5' region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S' region, could be responsible for dynamic events of gene rearrangement during evolution.

Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S′ and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS′-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5′ region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S′ region, could be responsible for dynamic events of gene rearrangement during evolution.

Effect of IFN-gamma receptor gene deletion on vaccinia virus virulence.

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.

Seven new mutations in the nicotinamide adenine dinucleotide reduced–cytochrome b5 reductase gene leading to methemoglobinemia type I

AbstractCytochrome b5 reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.

Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene (B8R) are attenuated In vivo without a concomitant reduction in immunogenicity.

The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-gamma) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-gamma, including human and rat IFN-gamma; it does not, however, bind significantly to murine IFN-gamma. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-gamma and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.

A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia

AbstractRecessive congenital methemoglobinemia due to nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription– polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned PCR products. The results showed that the propositus was homozygous for a G→A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr). To characterize the mutant enzyme, both glutathione S-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed in Escherichia coli and affinity purified. The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin. These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.

A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia

Recessive congenital methemoglobinemia due to nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription– polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned PCR products. The results showed that the propositus was homozygous for a G→A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr). To characterize the mutant enzyme, both glutathione S-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed in Escherichia coli and affinity purified. The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin. These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.

The Effects of Targeting the Vaccinia Virus B5R Protein to the Endoplasmic Reticulum on Virus Morphogenesis and Dissemination

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vΔB5R. Similar levels of B5R protein were made by each virus, but the B5R-K2X2 protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K2X4 protein colocalised with 1,4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K2X2 was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K2X2 were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K2X2 EEV.

Neutralizing and Protective Antibodies Directed against Vaccinia Virus Envelope Antigens

The infection mechanism of vaccinia virus is largely unknown. Neither the attachment protein of extracellular enveloped virus (EEV), the biologically relevant infectious form of the virus, nor its cellular receptor has been identified. Surprisingly, all former attempts using antibodies to block EEV infection of cellsin vitrohad failed. Here, we report the production of an anti-envelope hyperimmune serum with EEV neutralizing activity and show that a polyclonal antiserum against the extraviral domain of protein B5R also inhibited EEV infection.In vivo,mice vaccinated with B5R protein were protected against a lethal vaccinia virus challenge. This protectivity is likely to be mediated by neutralizing antibodies. Protein A33R, but not A34R and A36R, also proved to be protective in active and passive vaccination experiments. However, in contrast to B5R, A33R protectivity did not correlate with antibody titers. Because anti-A33R antibodies did not neutralize EEVin vitro,the protectivity mediated by A33R protein probably involves a mechanism different from simple antibody binding. Taken together, our results suggest that antibodies to a specific protective epitope or epitopes on protein B5R are able to prevent EEV infection. The protein encoded by the B5R gene is therefore likely to play a crucial role in the initial steps of vaccinia virus infection—binding to a host cell and entry into its cytoplasm.

Functional Analysis of Vaccinia Virus B5R Protein: Role of the Cytoplasmic Tail

Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread bothin vitroandin vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene.

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.