Identification and functional analysis of sequence variants in the long control region and the E2 open reading frame of bovine papillomavirus type 1 isolated from equine sarcoids

BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the variant LCR in the presence of the E2 variant was similar to the reference/wild-type sequences in equine cells, whereas in bovine cells the variant function was reduced by 50%. These data suggest the viral BPV variants commonly detected in sarcoids have an enhanced function in equine cells compared to their function in bovine cells.     

Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens

Summary.  Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer [4]. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process. Received January 24, 2000/Accepted May 30, 2000     

Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells

Virus Genes - The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an...     

Integration of human papillomavirus type 16 into cellular DNA of cervical carcinoma: preferential deletion of the E2 gene and invariable retention of the long control region and the E6/E7 open reading frames

The integration patterns of human papillomavirus (HPV) type 16 in the cellular DNA of six cervical carcinoma samples were analyzed by the Southern blot procedure. None of the HPV integrants retained the entire viral genome. Double HPV integration was found in one case while all other cases were single integrants. In some samples, internal deletion and selective amplification of the viral sequences were observed. On integration, the E2 open reading frame (ORF) was invariably lost but the E6/E7 ORFs and the long control region of the HPV-16 genome were retained in all seven integrations analyzed and may play a role in cellular transformation and/or maintenance of the transformed phenotype.     

Identification of novel E6-E7 sequence variants of human papillomavirus 16

The rate of evolution of the human papillomavirus 16 (HPV16) genome is low. However, the ability of the E6 oncoprotein to interact with distinct p53 variants causes selective pressure on the E6 gene. In addition, intratypic recombination events in the HPV16 E6 and E7 genes have been characterized as extraordinary phenomena during the evolutionary history of virus. In the present study, we identified two new sequence variants through nucleotide analysis of the E6-E7 region of the HPV16 genome. Maximum-likelihood and empirical Bayesian methods were used in order to identify positive selection at particular residues of the E6 and E7 genes. Using the single recombination breakpoint (SBP) method, we found evidence of recombination events in the E6 ORF. Nucleotide sequence analysis showed that the new sequence variants are phylogenetically distant from the other members of the population. Our results indicate that new evolutionary intermediates of HPV16 might be formed either though positive selective pressure or through recombination events by multiple infections with distinct HPV16 variants.     

Lineage analysis of human papillomavirus type 18 based on E6 region in cervical samples of Iranian women

Distinct human papillomavirus (HPV) 18 variants are thought to differ in oncogenic potential and geographic distribution. As such, understanding the regional variants of HPV 18 would be of great importance for evolutionary, epidemiological, and biological analysis. In this regard, the sequence variations of E6 gene were investigated to characterize more common variants of HPV 18 in normal cells, premalignant, and malignant samples collected from the cervix. In total, 99 samples of HPV 18 were analyzed by polymerase chain reaction and sequencing. In overall, lineages A was identified in all study subjects, among which sublineage A4 was dominant although the difference observed was not statistically significant with regard to different stages of disease. Sublineage A4 comprised 90.9% of samples and the remaining were belonged to sublineages A1, A2, A3, and A5 at the frequency of 6.1%, 1%, 1%, and 1%, respectively. In conclusion, our findings clearly highlight the sublineage A4 of HPV 18 as the most dominant variant in Iran.     

Molecular variants of human papillomavirus type 16 and 18 and risk for cervical neoplasia in Portugal

Persistent high-risk human papillomavirus (HPV) infection is considered as the central cause of invasive cervical cancer. Specific HPV 16 and 18 sequence variations were associated with an increased risk for progression. The purpose of this study was to analyze intratypic variations of HPV 16 and 18 within the E6 gene, MY09/11 and LCR regions, and to evaluate the risk of these variants for cervical neoplasia among Portuguese women. Cervical samples from 187 HPV 16-positive and 41 HPV 18-positive women with normal epithelium, cervical intraepithelial neoplasia, or invasive cervical cancer were amplified by type-specific PCR, followed by sequence and phylogenetic analysis. Sixteen new HPV 16 and 18 patterns are described in this paper. European HPV 16 variants were the most frequent (74.3%), particularly Ep-T350 (44.4%), followed by African (16.1%), and Asian-American (9.6%). Non-European HPV 16 variants were more frequent in pre-invasive lesions than in normal tissue and low-grade lesions. However, when analyzed separately, only African variants were associated significantly with an increased risk for cervical cancer. For HPV 18, the AsAi variant showed a trend, which was not statistically significant to an enhanced oncogenicity. European variants seemed to be significantly associated with a lower risk for cervical cancer development. The distribution of HPV 16 and 18 variants was not related to age or race among women living in the same geographical region. Knowledge of variants will be important for risk determination as well as for designing primers or probes for HPV detection methods, and for appropriate cervical cancer prevention strategies.     

Nucleotide and amino acid sequence variation in the L1 and E7 open reading frames of human papillomavirus type 6 and type 16.

Human papillomavirus (HPV) type 6 and type 16 DNA sequence variants were found by partially sequencing the L1 and E7 open reading frames, using templates generated with the polymerase chain reaction. Identical variants were found in patients from widely separated locations, such as the United States, the Philippines, and India. The same sequence variants of HPV 16 were found in women with invasive cervical carcinoma and in women with no evidence of disease. Variation in the predicted amino acid sequences of the HPV 16 L1 and E7 proteins was found. A single nucleotide change at position 6433 was found in about 50% of the HPV 16 DNAs, resulting in a change in predicted amino acid sequence from threonine to alanine at the equivalent position in the L1 protein. Predicted amino acid changes were found in the HPV 16 E7 proteins at amino acid positions 28, 29, and 47. Variation at these positions could affect known properties of the E7 protein, including binding to the retinoblastoma protein.     

The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.     

Sequence variation in the L1 gene of human papillomavirus type 16 from Africa

Summary A 297 bp fragment of the HPV 16 L1 gene was sequenced from 35 isolates originating from normal cervical scrapes, cervical intraepithelial neoplasia biopsies and cervical carcinoma biopsies from South African women. A total of six point mutations that differed from the prototype HPV 16 sequence were detected in various combinations, giving rise to six distinct types of variants. Only one of the isolates had a sequence identical to the prototype. Our results indicate that the HPV L1 gene is conserved and there is no evidence that specific viral HPV L1 variants are associated with specific pathology.