R. P. Minnaar

Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11.

Previous results indicated that SV40 small t is essential for SV40‐induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del‐11 cells). From these results we concluded that del‐11 cells contain a cellular ‘SV40 small t‐like’ factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative ‘small t‐like’ factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del‐11 but not in diploid cells and is able to trans‐activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans‐activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A‐mediated ability of SV40 small t to trans‐activate the HPV16 LCR.

Heat-shock induction of the human immunodeficiency virus long terminal repeat.

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.

Transcriptional activation of the major immediate early transcription unit of human cytomegalovirus by heat-shock, arsenite and protein synthesis inhibitors

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.

Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent.

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Modulation of the human papillomavirus type 16 induced transformation and transcription by deletion of loci on the short arm of human chromosome 11 can be mimicked by SV40 small t

The human papillomavirus (HPV) type 16 enhancer-promoter has been shown to be active in human fibroblasts with a deletion on the short arm of one chromosome 11 (karyotype 46,del(11)(p11.11p15.1)) but is virtually inactive in diploid human fibroblasts (Smits, Smits, Jebbink, and ter Schegget, 1990b, Virology, 176, 158-165). In diploid human embryonic fibroblasts, activation of the HPV16 enhancer-promoter could be achieved by expression of the SV40 small t. By cotransfecting SV40 small t cDNA together with HPV16 DNA into diploid cells, it was possible to increase the transforming activity of HPV16 by 10- 15-fold. Furthermore, SV40 small t was essential for the SV40 large T-induced morphological transformation of human diploid fibroblasts, whereas SV40 small t was dispensable for transformation of del-11 cells. We propose that, as a result of the deletion of loci on the short arm of chromosome 11 in del-11 cells, functions are expressed that mimic those of SV40 small t in transformation and trans-activation.

Regulation of human papillomavirus type 16 (HPV-16) transcription by loci on the short arm of chromosome 11 is mediated by the TATAAAA motif of the HPV-16 promoter.

The human papillomavirus type 16 (HPV-16) enhancer-promoter is virtually inactive in normal human diploid fibroblasts, but active in human fibroblasts with a deletion in the short arm of one chromosome 11 (del-11 cells). Since the HPV-16 enhancer with the simian virus 40 promoter is active in both cell types, the target for chromosome 11-regulated HPV-expression is likely to be located in the HPV-16 early promoter region (nucleotides 57 to 112). We show here that DNA-protein complexes formed with an HPV-16 promoter fragment are quantitatively different in del-11 cell and diploid cell extracts. This quantitative difference detected in band shift experiments disappeared upon mutation of the HPV-16 TATAAAA box to TATTTAT. This mutation also strongly reduced the activity of the HPV-16 enhancer-promoter in del-11 cells. These results indicate that TATA-binding proteins are involved in the chromosome 11-mediated regulation of HPV-16 gene expression.

Evaluation of two (semi-)nested VP1 based-PCRs for typing enteroviruses directly from cerebral spinal fluid samples

Human enteroviruses (EVs) are the leading cause of CNS-associated disease in childhood. Identification of the EV types that patients are infected with is essential for monitoring outbreaks, the emergence of new types or variants, epidemiological surveillance and contributes to patient management. Rapid and sensitive molecular detection methods are frequently used to detect EVs/HPeVs directly from CSF. This requires that sensitive EV typing methods from CSF material need to be developed. In the present study two nested PCR-based typing assays were evaluated. The performance of the EV-A and -B specific nested PCR protocol and the Codehop-based PCR protocol were analyzed with several TCID(50)-titrated EV-A to D strains and 22 EV positive CSF samples. The EV-A and -B protocol was found to be more sensitive than the Codehop protocol. The Codehop protocol showed a high degree of aspecific amplification products when run on a gel, and required additional gel purification. The detection limit of the two protocols varied between the types, ranging from 0.1TCID(50)/mL sample to 10(6)TCID(50)/mL sample. From the 22 EV positive CSF samples, 15 (68%) samples were typed using either protocol. All samples were characterized as members of species B (E30 (9), CAV9 (2), E6 (1), E11 (1), E21 (1), E25 (1)). Three samples (E30 (2) and E25 (1)) could only be typed using the EV-B protocol. In this study, selected EV strains could be typed using both assays at low virus concentrations, typically found in CSF. However, the EV-A and -B protocol was more sensitive than the Codehop protocol for primary typing of CSF samples.

Resistance to methylation de novo of the human cytomegalovirus immediate early enhancer in a model for virus latency and reactivation in vitro

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.