Exploring the roles of HPV16 variants in head and neck squamous cell carcinoma: current challenges and opportunities

The incidence of squamous cell carcinomas of the head and neck (HNSCC) is consistently increasing, in association with human papillomavirus (HPV) infection, especially HPV16. HPV variants show heterogeneity in the pathogenicity of cervical cancer, but little has been established about their relevance on HNSCC. This review addresses the distribution of HPV16 variants in HNSCC and their potential contribution to clinical practice. A search was performed in PubMed using the keywords HNSCC HPV16 variants. Sixty articles were identified between 2000 and 2020 and 9 articles were selected for a systematic analysis. Clinical cohorts comprised 4 to 253 patients aged between 17 and 91 years with confirmed HPV16-positive HNSCC. Samples were collected from fresh biopsies of the tumour, oral rinse or formol fixed/paraffin embedded tissue, from the oral cavity, oropharynx, hypopharynx, larynx and Waldeyer's tonsillar ring. HPV16 variants were identified using Sanger sequencing techniques. Seven studies addressed the HPV16 E6 gene, one studied E6 and E7, another studied L1 and one focused on the long control region. European variants represent 25–95%, Asian-American 5–57% and African 2–4% of the total isolates, suggesting a marked predominance of European strains. No correlations could be drawn with patient prognosis, partly because many studies relied on small patient cohorts. Additional studies are needed, particularly those employing next generation sequencing techniques (NGS), which will allow faster and accurate analysis of large numbers of samples.

Complete genome sequence of a HPV31 isolate from laryngeal squamous cell carcinoma and biological consequences for p97 promoter activity

Human papillomavirus type 31, although detected less frequently than HPV types 16 and 18, is associated with head and neck squamous cell carcinomas. Previous studies suggest that polymorphisms in the long control region (LCR) may alter the oncogenic potential of the virus. This study reports the first complete genome of a South African HPV31 isolate from a laryngeal squamous cell carcinoma. Sequence variations relative to the HPV31 prototype sequence were identified. The pBlue-Topo® vector, a reporter gene system was used to investigate the possible influence of these variations on the LCR promoter activity in vitro. Using mutagenesis to create two different fragments, β-galactosidase assays were used to monitor the effect of nucleotide variations on the p97 promoter. Increased β-galactosidase expression was observed in mutants when compared to the South African HPV31 LCR isolate. Enhanced transcriptional activity was observed with the mutant that possessed a single nucleotide change within the YY1 transcription factor binding site. In conclusion, sequence variation within the LCR of HPV31 isolates may have a functional effect on viral p97 promoter activity.

Molecular Characterization of Human Papillomavirus Type 159 (HPV159)

Human papillomavirus type 159 (HPV159) was identified in an anal swab sample and preliminarily genetically characterized by our group in 2012. Here we present a detailed molecular in silico analysis that showed that the HPV159 viral genome is 7443 bp in length and divided into five early and two late genes, with conserved functional domains and motifs, and a non-coding long control region (LCR) with significant regulatory sequences that allow the virus to complete its life cycle and infect novel host cells. HPV159, clustering into the cutaneotropic Betapapillomavirus (Beta-PV) genus, is phylogenetically most similar to HPV9, forming an individual phylogenetic group in the viral species Beta-2. After testing a large representative collection of clinical samples with HPV159 type-specific RT-PCR, in addition to the anal canal from which the first HPV159 isolate was obtained, HPV159 was further detected in other muco-cutaneous (4/181, 2.2%), mucosal (22/764, 2.9%), and cutaneous (14/554, 2.5%) clinical samples, suggesting its extensive tissue tropism. However, because very low HPV159 viral loads were estimated in the majority of positive samples, it seemed that HPV159 mainly caused clinically insignificant infections of the skin and mucosa. Using newly developed, highly sensitive HPV159-specific nested PCRs, two additional HPV159 LCR viral variants were identified. Nevertheless, all HPV159 mutations were demonstrated outside important functional domains of the LCR, suggesting that the HPV159 viral variants were most probably not pathogenically different. This complete molecular characterization of HPV159 enhances our knowledge of the genome characteristics, tissue tropism, and phylogenetic diversity of Beta-PVs that infect humans.

Nanog, in Cooperation with AP1, Increases the Expression of E6/E7 Oncogenes from HPV Types 16/18

Persistent infections with some types of human papillomavirus (HPV) constitute the major etiological factor for cervical cancer development. Nanog, a stem cell transcription factor has been shown to increase during cancer progression. We wanted to determine whether Nanog could modulate transcription of E6 and E7 oncogenes. We used luciferase reporters under the regulation of the long control region (LCR) of HPV types 16 and 18 (HPV16/18) and performed RT-qPCR. We found that Nanog increases activity of both viral regulatory regions and elevates endogenous E6/E7 mRNA levels in cervical cancer-derived cells. We demonstrated by in vitro mutagenesis that changes at Nanog-binding sites found in the HPV18 LCR significantly inhibit transcriptional activation. Chromatin immunoprecipitation (ChIP) assays showed that Nanog binds in vivo to the HPV18 LCR, and its overexpression increases its binding as well as that of c-Jun. Surprisingly, we observed that mutation of AP1-binding sites also affect Nanog’s ability to activate transcription, suggesting cooperation between the two factors. We searched for putative Nanog-binding sites in the LCR of several HPVs and surprisingly found them only in those types associated with cancer development. Our study shows, for the first time, a role for Nanog in the regulation of E6/E7 transcription of HPV16/18.

Lineage analysis of human papillomavirus type 39 in cervical samples of Iranian women

Background The data with regards to the regional variants of distinct HPV types is of great value. Accordance with this, this study aimed to investigate the sequence variations of E6 gene and long control region of HPV 39 among normal, premalignant and malignant cervical samples in order to characterize the frequent HPV 39 variants circulating in Tehran, Iran. Methods In total, 70 cervical samples (45 normal, 16 premalignant, and 9 malignant samples) infected with HPV 39 were analyzed by nested-PCR and sequencing. Results Our results revealed that all samples belonged to A lineage. Almost all sequences (98.6%) were classified in A1 sublineage and only one sample (1.4%) was A2 sub lineage. Conclusions Our findings showed that lineages A, sublineage A1, is dominant in Tehran, Iran. However, the small sample size was the most important limitations of this study. Further studies with larger sample size from different geographical regions of Iran are necessary to estimate the pathogenicity risk of HPV 39 variants in this population.

Variants in the Mitochondrial Genome Sequence of Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrycidae)

The lesser grain borer, Rhyzopertha dominica, is a coleopteran pest of stored grains and is mainly controlled by phosphine fumigation, but the increase in phosphine-resistant populations threatens efficacy. Some phosphine-resistant insects have reduced respiration, and thus studying the mitochondrial genome may provide additional information regarding resistance. Genomic DNA from an inbred laboratory strain of R. dominica was extracted and sequenced with both short (Illumina) and long (Pacific Biosciences) read technologies for whole genome sequence assembly and annotation. Short read sequences were assembled and annotated by open software to identify mitochondrial sequences, and the assembled sequence was manually annotated and verified by long read sequences. The mitochondrial genome sequence for R. dominica had a total length of 15,724 bp and encoded 22 trna genes, 2 rRNA genes, 13 protein coding genes (7 nad subunits, 3 cox, 2 atp, and 1 cytB), flanked by a long control region. We compared our predicted mitochondrial genome to that of another from a R. dominica strain from Jingziguan (China). While there was mostly agreement between the two assemblies, key differences will be further examined to determine if mutations in populations are related to insecticide control pressure, mainly that of phosphine. Differences in sequence data, assembly, and annotation also may result in different genome interpretations.

The complete mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) based on long-read nanopore sequencing

Background The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a severe and invasive pest of coconut and other palms throughout Asia and the Pacific. The biocontrol agent, Oryctes rhinoceros nudivirus (OrNV), has successfully suppressed O. rhinoceros populations for decades but new CRB invasions started appearing after 2007. A single-SNP variant within the mitochondrial cox1 gene is used to distinguish the recently-invading CRB-G lineage from other haplotypes, but the lack of mitogenome sequence for this species hinders further development of a molecular toolset for biosecurity and management programmes against CRB. Here we report the complete circular sequence and annotation for CRB mitogenome, generated to support such efforts. Methods Sequencing data were generated using long-read Nanopore technology from genomic DNA isolated from a CRB-G female. The mitogenome was assembled with Flye v.2.5, using the short-read Illumina sequences to remove homopolymers with Pilon, and annotated with MITOS. Independently-generated transcriptome data were used to assess the O. rhinoceros mitogenome annotation and transcription. The aligned sequences of 13 protein-coding genes (PCGs) (with degenerate third codon position) from O. rhinoceros, 13 other Scarabaeidae taxa and two outgroup taxa were used for the phylogenetic reconstruction with the Maximum likelihood (ML) approach in IQ-TREE and Bayesian (BI) approach in MrBayes. Results The complete circular mitogenome of O. rhinoceros is 20,898 bp in length, with a gene content canonical for insects (13 PCGs, two rRNA genes, and 22 tRNA genes), as well as one structural variation (rearrangement of trnQ and trnI) and a long control region (6,204 bp). Transcription was detected across all 37 genes, and interestingly, within three domains in the control region. ML and BI phylogenies had the same topology, correctly grouping O. rhinoceros with one other Dynastinae taxon, and recovering the previously reported relationship among lineages in the Scarabaeidae. In silico PCR-RFLP analysis recovered the correct fragment set that is diagnostic for the CRB-G haplogroup. These results validate the high-quality of the O. rhinoceros mitogenome sequence and annotation.

Genetic variability of the HPV16 early genes and LCR. Present and future perspectives

Human papillomavirus 16 (HPV16) infection is the aetiologic factor for the development of cervical dysplasia and is regarded as highly carcinogen, because it is implicated in more than 50% of cervical cancer cases, worldwide. The tumourigenic potential of HPV16 has triggered the extensive sequence analysis of viral genome in order to identify nucleotide variations and amino acid substitutions that influence viral oncogenicity and subsequently the initiation and progression of cervical cancer. Nowadays, specific mutations of HPV16 DNA have been associated with an increased risk of high-grade squamous intraepithelial lesions and invasive cervical cancer (ICC) development, including E6 : Q14H, H78Y, L83V, Ε7 : N29S, S63F, E2 : H35Q, P219S, T310K, E5 : I65V, whereas highly conserved regions of viral DNA have been extensively characterised. In addition, numerous novel HPV16 mutations are observed among the studied populations from various geographic regions, hence advocating that different HPV16 strains seem to emerge with different tumourigenic capacities. The present review focuses on the variability of the early genes and the long control region, emphasising on the association of specific mutations with the development of severe dysplasia. Finally, it evaluates whether specific regions of HPV16 DNA are able to serve as valuable biomarkers for cervical cancer risk.