Early immunity induced by a glycoprotein E-negative vaccine for infectious bovine rhinotracheitis.

Four groups of calves were vaccinated with a glycoprotein E-negative vaccine for infectious bovine rhinotracheitis. Two groups of calves were vaccinated intramuscularly and challenged with a wild-type virus 14 and seven days after being vaccinated. The other two groups were vaccinated intranasally and similarly challenged after four and three days; an unvaccinated control group was also challenged. All four vaccination schedules reduced the incidence of clinical signs and the excretion of wild-type virus, and these reductions occurred as early as three days after the intranasal vaccination even in the absence of neutralising antibodies. Because of its marker characteristics, vaccination with this vaccine would not interfere with the detection of infected cattle during an outbreak, and it should therefore provide a useful tool for emergency vaccination campaigns.

Fusion of the green fluorescent protein to amino acids 1 to 71 of bovine respiratory syncytial virus glycoprotein G directs the hybrid polypeptide as a class II membrane protein into the envelope of recombinant bovine herpesvirus-1.

It was recently shown that the class II membrane glycoprotein G of bovine respiratory syncytial virus (BRSV) is integrated into the envelope of recombinant bovine herpesvirus-1 (BHV-1) virions in the correct orientation. To verify the hypothesis that the membrane anchor of BRSV G might be suitable to target heterologous polypeptides into the membrane of recombinant BHV-1 particles, an open reading frame encoding a fusion protein between amino acids 1 to 71 of the BRSV G glycoprotein and the green fluorescent protein (TMIIGFP) was recombined into the genome of BHV-1. The resulting recombinant BHV-1/eTMIIGFP had growth properties similar to those of wild-type BHV-1. Live-cell analysis of cells infected with BHV-1/eTMIIGFP indicated that the fusion protein localized to the cell surface. Immunoprecipitations and virus neutralization assays using a GFP-specific antiserum proved that TMIIGFP was incorporated as a class II membrane protein into virions.

Susceptibility of bovine antigen-presenting cells to infection by bovine herpesvirus 1 and in vitro presentation to T cells: two independent events.

The aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4(+) T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2(+) or CD4(+)) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD-/-, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD-/- mutant virus could be used to induce virus-specific immune responses in cattle.

Bovine herpesvirus 1 requires glycoprotein H for infectivity and direct spreading and glycoproteins gH(W450) and gB for glycoprotein D-independent cell-to-cell spread.

By analogy with glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), gH may also be essential for penetration and cell-to-cell spread of bovine herpes-virus 1 (BHV-1). This was verified with a gH-negative BHV-1 mutant (gH-BHV-1), which replicated normally on gH-expressing cells but was unable to form plaques and infectious progeny on non-complementing cells. The block in entry could be overcome by polyethylene glycol-induced membrane fusion, demonstrating that gH is not essential for egress. Propagation of gH-BHV-1 on cell lines expressing wild-type gH or gH(W450), which complements the function of BHV-1 gD for cell-to-cell spread, indicated that gH(W450) is more efficient than wild-type gH in mediating direct spread of BHV-1. This was supported by the plaque sizes induced by rescued gH-BHV-1 that expressed wild-type gH and gH(W450). Infection of cell lines expressing gH of BHV-1, HSV-1 and PRV with gH-BHV-1, HSV-1 and PRV mutants demonstrated that heterologous gH molecules could not complement gH function in penetration or cell-to-cell spread.

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene.

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

Comparison of DNA application methods to reduce BRSV shedding in cattle.

We compared the protection afforded by three different DNA application methods against bovine respiratory syncytial virus (BRSV) infection in cattle. A synthetic gene that codes for the G protein of BRSV was inserted into a eukaryotic vector and was used in the vaccine. Intradermal (i.d.) application with a needleless injector (NI), the Pigjet, reduced BRSV excretion significantly better after BRSV challenge than intramuscular (i.m.) or i.d. vaccination with a needle. Serum antibodies against the G protein were consistently the highest and showed less variation in Calves vaccinated with the NI compared with those in i.m. and i.d. vaccinated calves. After BRSV challenge, secondary serum and mucosal antibody responses were also the highest in NI vaccinated calves. We conclude that DNA application with the needleless injector is substantially better than i.m. or i.d. application, and is capable to prime the immune response at the respiratory mucosa.

The class II membrane glycoprotein G of bovine respiratory syncytial virus, expressed from a synthetic open reading frame, is incorporated into virions of recombinant bovine herpesvirus 1.

The bovine herpesvirus 1 (BHV-1) recombinants BHV-1/eG(ori) and BHV-1/eG(syn) were isolated after insertion of expression cassettes which contained either a genomic RNA-derived cDNA fragment (BHV-1/eG(ori)) or a modified, chemically synthesized open reading frame (ORF) (BHV-1/eG(syn)), which both encode the attachment glycoprotein G of bovine respiratory syncytial virus (BRSV), a class II membrane glycoprotein. Northern blot analyses and nuclear runoff transcription experiments indicated that transcripts encompassing the authentic BRSV G ORF were unstable in the nucleus of BHV-1/eG(ori)-infected cells. In contrast, high levels of BRSV G RNA were detected in BHV-1/eG(syn)-infected cells. Immunoblots showed that the BHV-1/eG(syn)-expressed BRSV G glycoprotein contains N- and O-linked carbohydrates and that it is incorporated into the membrane of infected cells and into the envelope of BHV-1/eG(syn) virions. The latter was also demonstrated by neutralization of BHV-1/eG(syn) infectivity by monoclonal antibodies or polyclonal anti-BRSV G antisera and complement. Our results show that expression of the BRSV G glycoprotein by BHV-1 was dependent on the modification of the BRSV G ORF and indicate that incorporation of class II membrane glycoproteins into BHV-1 virions does not necessarily require BHV-1-specific signals. This raises the possibility of targeting heterologous polypeptides to the viral envelope, which might enable the construction of BHV-1 recombinants with new biological properties and the development of improved BHV-1-based live and inactivated vector vaccines.

Characterization of the bovine herpesvirus 1 UL8 gene and gene products.

The bovine herpesvirus 1 (BHV-1) strain Schönböken UL8 gene and the 5' flanking region were sequenced. Comparison of the UL8 ORF with the previously reported UL8 ORF of BHV-1 strain Cooper revealed significant differences that were mainly due to three frame-shifted segments. Reanalysis of the Cooper sequence after isolation of the respective segments from genomic DNA by PCR did not confirm the discrepancies; on the contrary, our results indicate a high degree of sequence conservation between the UL8 proteins of different BHV-1 isolates. A monospecific antiserum, raised against a bacterially expressed TrpE-UL8 fusion protein, identified the 80 kDa apparent molecular mass UL8 polypeptide which is localized in the nucleus of infected cells. Analysis of transcripts and time-course studies demonstrated that the UL8 protein is translated from a delayed-early expressed 3.1 kb polyadenylated mRNA which initiates within the UL9 ORF.

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV.

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) (BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of gamma/delta+ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.

Antiviral activity of an extract from leaves of the tropical plant Acanthospermum hispidum.

Incubation of the alphaherpesviruses pseudorabiesvirus (PRV) and bovine herpesvirus 1 during infection of cell cultures with an extract prepared from the leaves of Acanthospermum hispidum impaired productive replication of these viruses in a concentration-dependent manner whereas propagation of classical swine fever virus, foot-and-mouth disease virus and vaccinia virus was not affected. The 50% inhibitory concentration for cell growth (IC50) was 107 +/- 5 microliters/ml, and the concentration reducing PRV yield by 1 log10 (90% effective concentration, EC90) was 8 +/- 3 microliters/ml. The selectivity index calculated as the IC50/EC90 ration was 13 +/- 4. Delineation of the mechanism of the antiviral activity demonstrated inhibition of alphaherpesvirus attachment to and, to a lesser extent, penetration into the cells. In contrast, viral gene expression was not inhibited by the extract when added after entry of virions into the target cells. Reduced antiviral activity of A.h. against PRV deletion mutants lacking glycoprotein C (gC) or glycoproteins gC, gE, gG and gI altogether indicated that gC alone and/or viral attachment complexes of which gC is a component constitute the target structures for A. hispidum.

From essential to beneficial: glycoprotein D loses importance for replication of bovine herpesvirus 1 in cell culture.

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1) has been shown to be an essential component of virions involved in virus entry. gD expression in infected cells is also required for direct cell-to-cell spread. Therefore, BHV-1 gD functions are identical in these aspects to those of herpes simplex virus 1 (HSV-1) gD. In contrast, the gD homolog of pseudorabies virus (PrV), although essential for penetration, is not necessary for direct cell-to-cell spread. Cocultivation of cells infected with phenotypically gD-complemented gD- mutant BHV-1/80-221 with noncomplementing cells resulted in the isolation of the cell-to-cell-spreading gD-negative mutant ctcs+BHV-1/80-221, which was present in the gD-null BIV-1 stocks. ctcs+BHV-1/80-221 could be propagated only by mixing infected with uninfected cells, and virions released into the culture medium were noninfectious. Marker rescue experiments revealed that a single point mutation in the first position of codon 450 of the glycoprotein H open reading frame, resulting in a glycine-to-tryptophan exchange, enabled complementation of the gD function for cell-to-cell spread. After about 40 continuous passages of ctcs+BHV-1/80-221-infected cells with noninfected cells, the plaque morphology in the cultures started to change from roundish to comet shaped. Cells from such plaques produced infectious gD- virus, named gD-infBHV-1, which entered cells much more slowly than wild-type BHV-1. In contrast, integration of the gD gene into the genomes of gD-infBHV-1 and ctcs+BHV-1/80-221 resulted in recombinants with accelerated penetration in comparison to wild-type virions. In summary, our results demonstrate that under selective conditions, the function of BHV-1 gD for direct cell-to-cell spread and entry into cells can be compensated for by mutations in other viral (glyco)proteins, leading to the hypothesis that gD is involved in formation of penetration-mediating complexes in the viral envelope of which gH is a component. Together with results for PrV, varicella-zoster virus, which lacks a gD homolog, and Marek's disease virus, whose gD homolog is not essential for infectivity, our data may open new insights into the evolution of alphaherpesviruses.

Identification and characterization of the bovine herpesvirus 5 US4 gene and gene products.

The BHV-5 strain N569 (BHV-5/N569) homolog to the BHV-1 US4 gene was sequenced and characterized. RNA analyses showed that a 1.8-kb mRNA which contains the BHV-5/N569 US4 open reading frame initiates 55 nucleotides upstream from the predicted translational start codon and terminates 17 nucleotides downstream from the consensus sequence for polyadenylation. Comparison of the deduced amino acid sequences of the predicted US4 encoded proteins of BHV-5/N569 and BHV-1 strain Schönböken (BHV-1/Schö) revealed 75% identity. An antiserum, raised in rabbits after infection with a BHV-5/N569 US4 ORF expressing recombinant vaccinia virus, specifically precipitated a 65-kDa protein and a diffusely migrating protein species with an apparent molecular mass between 90 and > 240 kDa from the supernatant of BHV-5/ N569 infected cells. Treatment of immmunprecipitated proteins with chondroitinase AC demonstrated that the latter contains glycosaminoglycans. The mobility of the BHV-5/N569 US4 gene products was identical to the BHV-1 US4 ORF encoded glycoprotein G (gG) and glycoproteoglycan G (gpgG; G. M. Keil, T. Engelhardt, A. Karger, and M. Enz. J. Virol. 70, 3032-3038, 1996) and were therefore named BHV-5 gG and BHV-5 gpgG. Immunoprecipitations with sera from BHV-1 infected cattle indicated a type-specific immune response to gG, since these sera failed to react with vaccinia virus-expressed gG-5 but recognized vaccinia virus-expressed gG-1.

Bovine interleukins 2 and 4 expressed in recombinant bovine herpesvirus 1 are biologically active secreted glycoproteins.

The open reading frames encoding bovine interleukin 2 (boIL-2) and bovine interleukin 4 (boIL-4) were integrated into the unique short segment of the genome of bovine herpesvirus 1 (BHV-1) and expressed under control of the murine cytomegalovirus (MCMV) immediate-early 1 (ie1) enhancer-promoter element or the MCMV early 1 (e1) promoter. Madin-Darby bovine kidney cells infected with the recombinant viruses secreted boIL-2 or boIL-4 into the culture medium. Secretion was inhibited by the presence of brefeldin A during the infection, indicating that export from the cells was dependent on a functional Golgi apparatus. Treatment of the secreted interleukins with N-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed boIL-2 from 22 kDa to 16 kDa and that of boIL-4 from 20 kDa to 13 kDa, which demonstrated that both cytokines contain N-linked oligosaccharides. Digestion with neuraminidase and O-glycosidase had no detectable effect on the apparent molecular masses, suggesting that BHV-1-expressed boIL-2 and boIL-4 are not, or only slightly, O-glycosylated. In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed boIL-2 and boIL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively. In conclusion, we show that boIL-2 and boIL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.

Bovine herpesvirus 1 U(s) open reading frame 4 encodes a glycoproteoglycan.

Sequence analysis of the short unique (Us) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the Us open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the Us ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the Us ORF3 and Us ORF4, whereas the 1.8-kb RNA constitutes the monocistronic Us ORF4 mRNA. To identify the predicted BHV-I gG, recombinant vaccinia virus expressing the Us ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61- and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the Us ORF4 deletion mutant BHV-l/gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N- and O-linked carbohydrates, and that the high-molecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan C (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-I gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino-glycosylation, that has not yet been described for a herpesvirus-encoded protein.

Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.

The proposed gene for VP1 of HAV encodes for a larger protein than that observed in HAV-infected cells and virions.

The termini of hepatitis A virus (HAV) mature proteins have been assigned mainly by their homology to other picornaviruses and their apparent electrophoretic mobility; the proposed coding sequence for VP1 is supposed to encompass 900 nucleotides from position 2208 to 3107 of the HAV genome. In order to further characterize this protein, we analyzed the in vitro-and in vivo-synthesized translation products of the putative VP1 gene. cDNA coding for full-length VP1 was cloned under the control of a T7 promoter in pTF7-5; the resulting plasmid (pTF7-5/VP1) was used for both synthesis of RNA to program rabbit reticulocyte lysates and construction of a recombinant vaccinia virus (rvv/T7-VP1). Immunoblot analysis and immunoprecipitation using antisera raised against a synthetic peptide corresponding to amino acids 13 of 33 of VP1 (13-33/VP1) led to identification of a 37-kDa protein in lysates of in vitro translated VP1 and rvv/T7-VP1-infected HFS cells, whereas a 33-kDa protein was detected with purified virions and in lysates of HAV-infected HFS cells. Because the antiserum used was directed against an amino-terminal part of VP1 and the amino terminus of VP1 is identified by sequence analysis, these results show that VP1 present in the HAV virions and infected cells is shorter than previously proposed and suggest that the real carboxy terminus of VP1 is approximately 40 amino acids upstream. In order to limit the possible carboxy-terminal sites in the predicted region, we investigated in vitro synthesized translation products of a set of constructs with C-termini ending at potential cleavage sites for viral proteases 3C. The construct containing the nucleotides from position 2208 to 3026 codes for a protein (1-273/VP1) which exhibits the same electrophoretic mobility as VP1 synthesized by HAV in vivo.

Unidirectional complementation between glycoprotein B homologues of pseudorabies virus and bovine herpesvirus 1 is determined by the carboxy-terminal part of the molecule.

The most highly conserved glycoproteins in herpesviruses, homologues of glycoprotein B (gB) of herpes simplex virus, have been shown to play essential roles in membrane fusion during penetration and direct cell-to-cell spread of herpes virions. In studies aimed at assessing whether sequence conservation is reflected in the conservation of functional properties, we previously showed that bovine herpesvirus 1 (BHV-1) gB was able to functionally complement a gB- PrV mutant. To analyse in detail the function of gB in BHV-1, and to be able to test for reciprocal complementation between pseudorabies virus (PrV) and BHV-1 gB, we isolated a gB- BHV-1 mutant on a cell line stably expressing BHV-1 gB. Functional analysis showed that BHV-1 gB was essential for penetration as well as for direct cell-to-cell spread of BHV-1, indicating similar functions for PrV and BHV-1 gB. However, PrV gB was unable to complement plaque formation, i.e. direct cell-to-cell spread, or penetration of gB-BHV-1 virions despite its incorporation into the virion envelope. Analysis of cell lines expressing chimeric gB molecules composed of PrV and BHV-1 gB showed that plaque formation of both gB- mutants was complemented when the carboxy-terminal half of the chimeric gB was derived from BHV-1 gB and the amino-terminal half from PrV gB. In the opposite case, unidirectional complementation occurred. Although the chimeric molecules were generally less efficient in complementing infectivity of free virions, a similar complementation pattern was observed. In summary, our data show a unidirectional pattern of transcomplementation between the gB glycoproteins of PrV and BHV-1. This indicates that these proteins are functionally related but not identical. The unidirectional transcomplementation pattern was determined by the provenance of the carboxy-terminal half in chimeric gB proteins indicating that regions which are important for gB function but differ between PrV and BHV-1 reside in this part of gB.

Analysis of bovine herpesvirus 4 genomic regions located outside the conserved gammaherpesvirus gene blocks.

Bovine herpesvirus 4 (BHV-4) DNA sequences located outside the gene blocks conserved among the gammaherpesviruses BHV-4, herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) were analysed. Twelve potential open reading frames (ORFs) were found. Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or betaherpesviruses. Nevertheless, six of the ORFs were homologous in amino acid sequences to proteins encoded by HVS but apparently not to those encoded by EBV. Furthermore, the location and orientation of these six ORFs in the BHV-4 genome were similar to the corresponding ORFs in the HVS genome. No genes homologous to known cellular genes were found in the BHV-4 genome; this feature is the major difference between the BHV-4 and HVS genomes with regards to the overall gene content.

Site-directed mutagenesis in the active site of the herpes simplex virus type 1 thymidine kinase gene.

The thymidine kinase (TK) of herpes simplex virus type 1 (HSV-1) contains three regions of homology to other ATP utilizing enzymes. We have altered one region of the protein, which seems to play an important role in phosphorylation substrates by site-directed mutagenesis. When the aspartate 162 was changed to asparagine, the enzyme lost its activity. To identify the inactive protein, expressed by a vaccinia vector in eukaryotic cells, a monospecific antiserum against a bacterial tryptophan E-HSV-1 TK fusion protein was made. These results support the suggestion that aspartate 162 is essential for the enzymatic activity.

Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog.

In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) collocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, Bühler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66, 4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51% (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the close relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection.