Richard R. Spaete

Human cytomegalovirus strain towne glycoprotein B is processed by proteolytic cleavage

The gene encoding glycoprotein B of human cytomegalovirus (CMV) strain Towne was cloned, sequenced, and expressed in order to study potential targets for viral neutralization. Secondary structure analysis of the 907 amino acid protein predicted a 24 amino acid N-terminal signal sequence and a potential transmembrane region composed of two domains, 34 and 21 amino acids. The CMV (Towne) gB gene had a 94% nucleotide similarity and a 95% amino acid similarity to the CMV (AD169) gB gene [as described by M.P. Cranage et al. (1986, EMBO J. 5, 3057-3063)]. Transcriptional analysis of the CMV (Towne) gB coding strand revealed that the gB message (3.9 kb), was transcribed from this region as early as 4 hr postinfection, and well in advance of gB protein synthesis. Full-length and truncated versions of the gB gene were expressed in COS cells using expression vectors where transcription was driven by the SV40 early promoter or the CMV major immediate early promoter. Expression was detected by immunofluorescence and ELISA using the virus neutralizing murine monoclonal antibody 15D8 (L. Rasmussen, J. Mullenax, R. Nelson, and T.C. Merigan, 1985, J. Virol. 55, 274-280). This antibody had been shown previously to recognize a 55-kDa CMV virion protein and a related 130-kDa intracellular precursor. Amino acid sequence analysis of the N-terminus of the 55-kDa viral glycoprotein (gp55) showed that gp55 is derived from gB (gp130) by proteolytic cleavage and represents the C-terminal region of gp130. The truncated version of gB expressed in COS and CHO cells was also processed by proteolytic cleavage as demonstrated by Western blotting. Our study localizes the epitope recognized by 15D8 to within a 186 amino acid fragment of the gp55 protein. These results indicate that CMV gB is a target for neutralization and establishes gp55 as a candidate component for use in a subunit vaccine.

Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells

Truncated and full-length versions of the hepatitis C virus protein domain encoding a presumptive envelope glycoprotein designated E2/NS1 were stably expressed in CHO cell lines. Characterization of the processing events involved in the maturation of E2/NS1 revealed that a high-mannose form resident in the endoplasmic reticulum was the most abundant form detected intracellularly. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that the E2/NS1 glycoprotein resided in the endoplasmic reticulum. The full-length form of E2/NS1 appeared to be cell-associated and could not be detected as a secreted product. C-terminal truncated molecules could be detected in the extracellular media as fully processed glycoproteins containing terminal sialic acid additions. These truncated glycoproteins are predicted to be biologically relevant targets of the host immune response and are therefore potential subunit vaccine candidates.

Human cytomegalovirus structural proteins.

Introduction. Members of the Herpesviridae family are viruses with a core containing the viral DNA in the form of a torus, a capsid (100 nm in diameter and composed of 162 capsomers), a tegument, which is a structure of variable thickness between the capsid and the envelope, and an envelope. The envelope is the trilaminar outer covering which contains numerous protrusions of spikes consisting of virally encoded glycoproteins. The resulting diameter of the virion varies from 120 to 300 nm. Herpesviruses have been divided into three subfamilies on the basis of biological properties. The Alphaherpesvirinae are herpesviruses with a variable host range, short replication cycle, rapid spread in cultured cells, a lytic effect on infected cells and a tropism for establishing a latent infection in sensory ganglia. The Betaherpesvirinae have a restricted host range, a longer replication cycle, an infection which progresses more slowly in cultured cells and a latent state that can be established in numerous cells and tissues.

Evaluation of a Live Attenuated Recombinant Virus RAV 9395 as a Herpes Simplex Virus Type 2 Vaccine in Guinea Pigs

Recombinant virus RAV 9395 was constructed by deleting both copies of the gamma(1)34.5 gene, and the UL55 and UL56 open reading frames from herpes simplex virus type 2 (HSV-2) strain G. The potential use of RAV 9395 as an HSV-2 vaccine was investigated by evaluating the ability of RAV 9395 to protect guinea pigs from severe disease by HSV-2(G) challenge. RAV 9395 administered intramuscularly reduced both lesion development and severity in a dose-dependent manner in guinea pigs challenged with HSV-2(G). The frequency of reactivation of RAV 9395 from explanted dorsal root ganglia was low compared with that of HSV-2(G). Immunization with RAV 9395 at doses of 1 x 10(5) pfu and above generally precluded the establishment of latency by the challenge virus. The results presented in this report lend support for the development of genetically engineered live HSV vaccines.

The human cytomegalovirus strain towne glycoprotein H gene encodes glycoprotein p86

The gene encoding the glycoprotein H (gH) homologue of CMV strain Towne was cloned, sequenced, and expressed. The predicted 742 amino acid gH protein had characteristics typical of a membrane glycoprotein including hydrophobic signal and transmembrane domains and six possible N-linked glycosylation sites. The CMV (Towne) gH gene had a 95% nucleotide identity and a 96.6% amino acid identity with the CMV (AD169) gH gene, as described by M. P. Cranage, G. L. Smith, S. E. Bell, H. Hart, C. Brown, A. T. Bankier, P. Tomlinson, B. G. Barrell, and T. C. Minson (1988, J. Virol. 62, 1416-1422). Transcriptional analysis of the gH gene revealed that the 2.9-kilobase (kb) gH transcript was not detected until late after CMV infection, indicating that the kinetics of gH expression were typical of the late class of CMV genes. The gH gene was expressed in COS cells using a vector in which transcription was driven by the SV40 early promoter. The expression of gH was detected by immunofluorescence using the virus neutralizing murine monoclonal antibody 1G6, which is specific for an 86-kilodalton (kDa) CMV virion membrane protein (p86). Amino acid sequence analysis of p86 tryptic peptides revealed sequence identity with peptides from the deduced gH amino acid sequence, confirming that the gH gene encodes p86. These results indicate that CMV gH can induce virus neutralizing antibodies and establishes gH as a candidate antigen for a subunit vaccine against CMV.

A Major Neutralizing Domain Maps within the Carboxyl-terminal Half of the Cleaved Cytomegalovirus B Glycoprotein

Cytomegalovirus (CMV) encodes several glycoproteins reported to be structural homologues of glycoproteins encoded by herpes simplex virus type 1 (HSV-1). To map the antigenic and functional domains on the 907 amino acid CMV glycoprotein B (gB), we cloned and expressed a subfragment of BamHI fragment R of the CMV (Towne) genome into an expression vector and reacted the resulting gene product with a panel of monoclonal antibodies. Our results showed that the DNA fragment encodes related glycoproteins which we previously designated gA and which others have reported to be homologous to HSV-1 gB in CMV (AD169). Analyses of the processing of CMV gB transiently expressed in eukaryotic cells showed that glycosylation occurred independently of viral infection. Ten antibodies with complement-dependent and independent neutralizing activity reacted with a truncated derivative of gB that contained 619 amino-terminal residues but lacked the transmembrane and intracellular regions of the molecule. Twelve additional antibodies reacted with a CHO cell line expressing a 680 amino-terminal derivative of gB. All of the reactive antibodies precipitated the 447 residue carboxy-terminal cleavage product of gB from extracts of CMV-infected cells. These results showed that the neutralizing epitopes map in at least two domains of gB which are located in a discontinuous segment of 219 amino acids between residues 461 and 680 from the amino terminus of the molecule.

Primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication.

Cytomegaloviruses generally display a host range restricted to differentiated cell types from the species they infect. For human cytomegalovirus (HCMV) this has meant that with few exceptions tissue culture systems have relied on the use of primary foreskin fibroblast (HF) cells or primary human embryonic lung cells to study gene expression and virus replication functions. We have observed that primary skin fibroblast (CF) cells derived from the chimpanzee (Pan troglodytes) support the replication of a laboratory strain (Towne) of HCMV. The kinetics of gene expression of the Towne strain grown in CF or HF cells appeared to be equivalent. Titres of progeny virions grown in CF cells appeared to be reduced 10-fold relative to those of virus grown in HF cells. In contrast, replication of the Towne virus was not supported by growth in WES cells (ATCC no. CRL 1609), a chimpanzee skin fibroblast cell line transformed by an adenovirus 12-simian virus 40 hybrid. This study shows that HCMV is less parochial in its host range than previously thought.