- Volume 77, Issue 6, 1996

Perinatal transmission of human papillomavirus type 16 (HPV-16) and persistence of virus DNA in infants until 6 months of age has been described. To confirm the origin of infant infections as maternal, we determined the nucleotide sequence of the upstream regulatory region (URR; bp 7540 to 157) of HPV-16 in samples from 13 HPV-16 DNA-positive mothers and their infants at 6 weeks and 2 years of age. Identical HPV-16 variant URR sequences were found in two mother/infant samples and similar variants were found in three sets. Four mothers with samples which contained prototypic HPV-16 sequences delivered infants who also had the prototypic sequence. Four mothers with variant URRs delivered infants who harboured either prototypic or different URR variants. Thus, concordant variants or prototypic sequences were detected in nine of 13 mother/infant samples, indicating that up to 69.2% of HPV-16-positive infants acquire virus from their mothers.

Previously, we have shown that E5a can induce ex pression of the c-jun gene in human papillomavirus (HPV)-11 E5a transformed NIH 3T3 cells and human epidermal keratinocytes. In this study, we investigated the relationship between expression of the E5a gene and c-jun in pathologically confirmed condylomata specimens using mRNA hybridization in situ. The c-jun RNA concentration was significantly higher in condylomata specimens with E5a mRNA expression than in specimens without E5a mRNA expression, or in normal cervical specimens. The cells with c-jun expression were located predominantly in the basal and parabasal cell layers. These layers were also the primary location of E5a-expressing cells. This is the first demonstration of a strong correlation (74%) between expression of the E5a and c-jun genes in condylomata specimens. This correlation might reflect regulation by HPV-11 E5a of c-jun gene expression in condyloma.

Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection. Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain. Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts. In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1. These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.

We have studied the activity of reporter plasmids, carrying the Epstein—Barr virus (EBV) nuclear antigen (EBNA) promoters Wp and Cp, in a group of somatic cell hybrids obtained by fusing EBV-positive lympho-blastoid cell lines or group II/III Burkitt’s lymphoma cell lines with non-B cell lines. In B/non-B cell hybrids of this type, B cell markers are extinguished as a rule, in parallel with the inactivation of Cp or Wp and down-regulation of EBNA-2-6 expression. A Wp-carrying reporter construct was active in non-B cell lines. Only cells with a B cell phenotype could support the activity of Cp-carrying plasmids. EBNA-2 transactivated Cp only in B cells. Our data suggest that while Wp can be used for EBNA transcription in B and non-B cells, Cp activity is restricted to B cells. The inability of EBNA-2 to transactivate Cp in non-B cells indicates that other factors present in B cells might be involved in Cp transactivation.

The Epstein—Barr virus (EBV) open reading frame (ORF) BCRF1, expressed in the late phase of the viral cycle, encodes a homologue of human interleukin-10 (hIL-10). Unspliced, 3′ co-terminal transcripts of 0.8 and 1.6 kb from O-tetradecanoylphorbol 13-acetate (TPA)-treated B95-8 cells have been described but other results indicated the existence of uncharacterized transcript(s) initiated upstream of the 1.6 kb BCRF1 mRNA. Here we describe two additional large transcripts of the BCRF1 ORF, a possibly spliced product of 3.5 kb and an unspliced product of 4.5 kb. The time course of the expression of BCRF1 transcripts and of the secreted protein from Akata cells were also determined.

Epstein—Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD). To determine whether the donor EBV isolate is transmitted to the recipient via the allograft and causes PTLD, EBV isolates from four cases of PTLD in cadaveric heart and/or lung transplant recipients were compared with the donor isolates by PCR and DNA sequence analysis. Two recipients who were EBV seronegative at transplantation acquired an EBV isolate indistinguishable from that of the donor and developed PTLD. In contrast, in two patients who were seropositive before transplantation, the donor isolate differed from that present in PTLD of the recipient. The results suggest that the acquisition of donor EBV is a risk factor for PTLD development in a previously seronegative transplant recipient.

Epstein—Barr virus (EBV) infection of the common marmoset causes long-term infection, with production of antibodies to virus-induced antigens, without clinical illness. Attempts to show the presence of EBV DNA in saliva of infected animals by PCR were initially unsuccessful, although slot-blot hybridization analysis demonstrated that viral DNA was present. Further investigations showed that most samples of pilocarpine-induced saliva, and 33% of the samples of whole mouth fluids (WMF) tested, were inhibitory to PCR. Similar results were found using human WMF. A method of assessing samples of marmoset WMF for the presence of EBV, by PCR using an EBV BamHI W probe, and removing inhibition with Chelex 100, is described. A total of 202 samples from 21 EBV infected, and seven non-infected animals was tested. Five seropositive animals shed virus on every occasion, and 15 intermittently. Two marmosets, infected as neonates, showed progressively increasing humoral responses to viral antigens, and shed virus on every occasion tested over 3 years. When mated with uninfected animals, the latter seroconverted 4 and 6 weeks later, respectively, and later shed virus into their WMF. The naturally infected animals were paired with naïve marmosets, and were able to pass on infection. These results establish that long-term, permissive EBV infection occurs in the common marmoset, and demonstrate again the similarities in the response to EBV between marmoset and man.