Lucy Rasmussen

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.

Human cytomegalovirus strains associated with congenital and perinatal infections.

The genotypes of human cytomegalovirus (HCMV) isolates from pediatric patients differs from those of infected adults in Australia. Genotypes were determined by PCR amplification of glycoprotein B (gB) sequences, with subsequent analysis by restriction fragment length polymorphism, single‐stranded conformation polymorphism, heteroduplex mobility analysis and direct DNA sequencing. Restriction fragment length polymorphism analysis of gB showed genotypes gB1 (39%) and gB3 (30%) were more prevalent in infected children and two new genotypes (gB6 and gB7) were found. Single‐stranded conformation polymorphism was used to group isolates into 22 further subtypes and suggested longitudinal co‐infection or viral mutation was occurring over time. Heteroduplex mobility analysis was found to be the most accurate and concise of the four methods used for genotyping HCMV isolates. DNA sequencing was used to confirm the results obtained from heteroduplex mobility analysis, and identified two isolates that were incorrectly genotyped by restriction fragment length polymorphism analysis. Heteroduplex mobility analysis efficiently genotyped all samples and allowed estimation of sequence variation between isolates. These data suggest certain gB genotypes are associated more commonly with childhood infections, and these differ from strains associated with invasive disease in HIV patients. J. Med. Virol. 61:481–487, 2000.

Cellular and Humoral Immunity in the Pathogenesis of Recurrent Herpes Viral Infections in Patients with Lymphoma

86 patients with lymphoma were evaluated prospectively for clinical and laboratory evidence of recurrent varicella-zoster, herpes simplex, and cytomegalovirus infections during the first 16 mo of treatment. Cellular immunity to the viral antigens was measured by in vitro lymphocyte transformation and interferon production. Antibody titers and nonspecific measures of cellular immunity, including T-cell quantitation and transformation to phytohemagglutinin, were also assessed. The patients treated with radiation and chemotherapy had the highest incidence of reactivation of each of the viruses (15-19%). Greater susceptibility to herpes viral reactivation in these patients correlated with suppression of cell-mediated immunity to the specific virus. In individual patients, suppression of cellular immunity to the specific herpes viral antigen preceded each episode of reactivation, but recurrent infection did not occur in all patients with diminished specific lymphocyte transformation. Absence of the response appears to be a necessary but not a sufficient condition for the recrudescence of latent infection. Better preservation of cellular immunity to herpes simplex antigen during treatment was associated with infrequent reactivation of herpes simplex. In 25 patients with acute herpes zoster, uncomplicated recovery from the infection was accompanied by the development of lymphocyte transformation and interferon production to varicella-zoster antigen. Quantitation of T-cell numbers and phytohemagglutinin transformation did not correlate with the presence of viral cellular immunity in treated patients. Responses returned while T-cell numbers were low, and the recovery of phytohemagglutinin transformation often preceded recovery of the responses to viral antigens. Although some patients had deficiencies in viral cellular immunity at diagnosis, the duration of the suppression of specific antiviral responses resulting from treatment appears to be the most important factor predisposing to the recurrence of herpes infections in lymphoma patients.

Inter- and Intragenic Variations Complicate the Molecular Epidemiology of Human Cytomegalovirus

Human cytomegalovirus isolates were analyzed, both by restriction fragment-length polymorphism typing and by sequencing for intra- and intergenic variability at 9 sites on the genome, to determine whether genetic variation influenced disease outcome and whether linkage among genes could be identified. Variation at the UL55 (glycoprotein B [gB]), UL74 (gO), UL75 (gH), UL115 (gL), US9, and US28 gene open-reading frames was studied in relationship to outcome of cytomegalovirus disease. Major findings were that (1) on the basis of analysis of only 9 genomic sites, it is apparent that an almost infinite number of genetic combinations are theoretically possible; (2) genetic linkages are rare; (3) intragenic variability may be a complicating factor in molecular epidemiologic studies; and (4) analysis of only a single gene from a clinical isolate may not reveal the presence of either intragenic variants or mixtures of genotypes.

The Genes Encoding the gCIII Complex of Human Cytomegalovirus Exist in Highly Diverse Combinations in Clinical Isolates

ABSTRACT The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.

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.

Characterization of two different human cytomegalovirus glycoproteins which are targets for virus neutralizing antibody

In previous studies we have identified two viral polypeptides detected by murine monoclonal antibodies which neutralize the infectivity of human cytomegalovirus (CMV) AD169. One is an 86,000-Da polypeptide (p86) and the second is a complex of two major coimmunoprecipitating polypeptides of 130,000 and 55,000 Da (p130/55). In this study we have shown that the two viral polypeptides are immunologically unrelated and have distinct peptide cleavage patterns. We have characterized these polypeptides as glycoproteins and studied their biosynthesis in human embryonic lung cells. The oligosaccharides found on both the p86 and the p130/55 were characterized by endoglycosidase digestion as N-linked high-mannose carbohydrates. Inhibitors of glycosylation were used to further characterize the oligosaccharides. Tunicamycin, which inhibits the biosynthesis of N-linked oligosaccharides on the endoplasmic reticulum, inhibited both the infectivity and biosynthesis of the p86 and p130/55. The underglycosylated forms in tunicamycin-treated cultures could be detected only under conditions of pulse-labeling with L-[35S]methionine. Monensin, which inhibits the modification of glycoproteins from simple to complex forms in the Golgi, reduced viral infectivity at concentrations which had no effect on viral protein synthesis, but did not alter the apparent molecular weight of either the p86 or the p130/55. The oligosaccharides were critical for the in vitro immunologic reactivity of the p86 in immunoblots. However, endoglycosidase F-treated p86 was comparable to the native form in inducing virus neutralizing antibody in guinea pigs. Endoglycosidase F-treated p130/55 retained its ability to bind antibody in Western blots.

Risk for Retinitis in Patients with AIDS Can Be Assessed by Quantitation of Threshold Levels of Cytomegalovirus DNA Burden in Blood

Cytomegalovirus (CMV) retinitis in patients infected with human immunodeficiency virus (HIV) is a significant clinical problem. Seventy-five patients with CD4 T cell counts <100/mm3 were monitored prospectively every 2 months for CMV DNA burden. The target for DNA amplification was a 162-bp fragment from the CMV immediate early gene. CMV DNA burden, at levels of > or =320 in white blood cells or > or =32 in plasma (P = .001), particularly when sustained (P = .005 and .008, respectively), distinguished patients who developed retinitis from those who remained free of disease. Progression to retinitis was not consistently accompanied by increases in CMV burden, indicating that quantitation of CMV burden beyond threshold levels is not necessary to predict risk for development of retinitis. Virus isolation from WBC, but not urine, was also significantly associated with risk for retinitis (P = .001).

Human cytomegalovirus polypeptides stimulate neutralizing antibody in vivo.

At least three human cytomegalovirus polypeptides are targets for virus neutralizing antibody; a single protein of 86,000 molecular weight (p86) and two coimmunoprecipitating proteins of 130,000 and 55,000 molecular weight (p130/55). These polypeptides have been isolated by immunoaffinity chromatography and tested for immunogenicity in guinea pigs. Neutralizing antibody was detected after immunization with both p86 and p130/55. Hyperimmune sera to p130/55, but not p86, were dependent upon guinea pig complement for virus neutralization.

Inhibition of human cytomegalovirus replication by 9-(1,3-dihydroxy-2-propoxymethyl)guanine alone and in combination with human interferons.

The inhibitory action of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on the replication of human cytomegalovirus was studied. Three laboratory strains (AD-169, Towne, and Davis) and three early passage (less than 10) clinical isolates were all inhibited in yield inhibition assays. In cultures infected with AD-169, virus yields could be inhibited if the drug was added as late as 3 days after the replication cycle had begun. The effects of the drug were fully reversible during the first 4 days of the viral replication cycle. Viral infectivity and viral DNA synthesis were reduced more than viral protein synthesis. Synergistic antiviral effects were observed with beta-cysteine, and to a lesser extent, with beta-serine recombinant interferons, but only over a narrow range of dose combinations. Images