Phylogenomic Analysis of Human Papillomavirus Type 31 and Cervical Carcinogenesis: A Study of 2093 Viral Genomes.

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17-2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

Genetic and Epigenetic Variations of HPV52 in Cervical Precancer.

The goal of this study was to identify human papillomavirus (HPV) type 52 genetic and epigenetic changes associated with high-grade cervical precancer and cancer. Patients were selected from the HPV Persistence and Progression (PaP) cohort, a cervical cancer screening program at Kaiser Permanente Northern California (KPNC). We performed a nested case-control study of 89 HPV52-positive women, including 50 cases with predominantly cervical intraepithelial neoplasia grade 3 (CIN3) and 39 controls without evidence of abnormalities. We conducted methylation analyses using Illumina sequencing and viral whole genome Sanger sequencing. Of the 24 CpG sites examined, increased methylation at CpG site 5615 in HPV52 L1 region was the most significantly associated with CIN3, with a difference in median methylation of 17.9% (odds ratio (OR) = 4.8, 95% confidence interval (CI) = 1.9-11.8) and an area under the curve of 0.73 (AUC; 95% CI = 0.62-0.83). Complete genomic sequencing of HPV52 isolates revealed associations between SNPs present in sublineage C2 and a higher risk of CIN3, with ORs ranging from 2.8 to 3.3. This study identified genetic and epigenetic HPV52 variants associated with high risk for cervical precancer, improving the potential for early diagnosis of cervical neoplasia caused by HPV52.

Cross-protection of the Bivalent Human Papillomavirus (HPV) Vaccine Against Variants of Genetically Related High-Risk HPV Infections.

BACKGROUND: Results from the Costa Rica Vaccine Trial (CVT) demonstrated partial cross-protection by the bivalent human papillomavirus (HPV) vaccine, which targets HPV-16 and HPV-18, against HPV-31, -33, and -45 infection and an increased incidence of HPV-51 infection. METHODS: A study nested within the CVT intention-to-treat cohort was designed to assess high-risk HPV variant lineage-specific vaccine efficacy (VE). The 2 main end points were (1) long-term incident infections persisting for ≥2 years and/or progression to high-grade squamous intraepithelial lesions (ie, cervical intraepithelial neoplasia grade 2/3 [CIN 2/3]) and (2) incident transient infections lasting for <2 years. For efficiency, incident infections due to HPV-16, -18, -31, -33, -35, -45, and -51 resulting in persistent infection and/or CIN 2/3 were matched (ratio, 1:2) to the more-frequent transient viral infections, by HPV type. Variant lineages were determined by sequencing the upstream regulatory region and/or E6 region. RESULTS: VEs against persistent or transient infections with HPV-16, -18, -33, -35, -45, and -51 did not differ significantly by variant lineage. As the possible exception, VEs against persistent infection and/or CIN 2/3 due to HPV-31 A/B and HPV-31C variants were -7.1% (95% confidence interval [CI], -33.9% to 0%) and 86.4% (95% CI, 65.1%-97.1%), respectively (P = .02 for test of equal VE). No difference in VE was observed by variant among transient HPV-31 infections (P = .68). CONCLUSIONS: Overall, sequence variation at the variant level does not appear to explain partial cross-protection by the bivalent HPV vaccine.

HPV16 CpG methyl-haplotypes are associated with cervix precancer and cancer in the Guanacaste natural history study.

OBJECTIVE: To evaluate HPV16 CpG methylation and methyl-haplotypes and their association with cervix precancer and cancer utilizing massively parallel single molecule next-generation sequencing (NGS). METHODS: A nested case-control study of HPV16 positive women was performed in a prospective cohort from Guanacaste, Costa Rica designed to study the natural history of HPV and cervical neoplasia. Controls encompassed 31 women with transient infections; there were 44 cases, including 31 women with CIN3 and 13 with cervical cancer. DNA samples from exfoliated cervical cells were treated with bisulfite and four regions (E6, E2, L2 and L1) were amplified with barcoded primers and tested by NGS. CpG methylation was quantified using a bioinformatics pipeline. RESULTS: Median methylation levels were significantly different between the CIN3+ cases versus controls in the E2, L2, and L1 regions. Methyl-haplotypes, specifically in 5 CpG sites included in the targeted L2 region, with the pattern "--+-+" had the highest Area Under the Curve value (AUC=88.40%) observed for CIN3 vs. CONTROLS: The most significant CpG site, L2 4277, determined by bisulfite NGS had an AUC=78.62%. CONCLUSIONS: This study demonstrates that NGS of bisulfite treated HPV DNA is a useful and efficient technique to survey methylation patterns in HPV16. This procedure provides quantitative information on both individual CpG sites and methyl-haplotypes that identify women with elevated present or subsequent risk for HPV16 CIN3 and cancer.

HPV16 methyl-haplotypes determined by a novel next-generation sequencing method are associated with cervical precancer.

We have developed and evaluated a next-generation bisulfite sequencing (NGS) assay to distinguish HPV16 cervical precancer (CIN2-3; N=59) from HPV16-positive transient infections (N=40). Cervical DNA was isolated and treated with bisulfite and HPV16 methylation was quantified by (i) amplification with barcoded primers and massively parallel single molecule sequencing and (ii) site-specific pyrosequencing. Assays were evaluated for agreement using intraclass correlation coefficients (ICC). Odds ratios (OR) for high methylation vs. low methylation were calculated. Single site pyrosequencing and NGS data were correlated (ICC=0.61) and both indicated hypermethylation was associated with precancer (ORs of 2-37). Concordant NGS and pyrosequencing results yieled ORs that were stronger when compared with using either assay separately. Within the L1 region, the ORs for CIN2-3 were 14.3 and 22.4 using pyrosequencing and NGS assays, respectively; when both methods agreed the OR was 153. NGS assays provide methylation haplotypes, termed methyl-haplotypes from single molecule reads: cases had increased methyl-haplotypes with ≥1 methylated CpG site(s) per fragment compared with controls, particularly in L1 (p=3.0×10(-8)). The maximum discrimination of cases from controls for a L1 methyl-haplotype had an AUC of 0.89 corresponding to a sensitivity of 92.5% and a specificity of 73.1%. The strengthening of the OR when the two assays were concordant suggests the true association of CpG methylation with precancer is stronger than with either assay. As cervical cancer prevention moves to DNA testing methods, DNA based biomarkers, such as HPV methylation could serve as a reflex strategy to identify women at high risk for cervix cancer.

Human papillomavirus genomics: past, present and future.

Human papillomaviruses (HPV) are a group of divergent DNA viruses, of which a select few evolutionarily related HPVs have emerged to be highly oncogenic and of significant medical importance. Essentially all cases of cervical cancer, as well as a subset of other anogenital and oral cancers are caused by this limited set of HPV types. At present, over 150 HPV types have been identified and may be classified into genera, species and types based upon comparison of the viral genome. Established nucleotide phylogenies sort the highly pathogenic HPV types to the genus Alphapapillomavirus (α-PV). A species group includes viral types with 60-70% genomic nucleotide similarity that share a most-recent common ancestor; for example the species group's alpha-9 (HPV16-related) and alpha-7 (HPV18-related), contain the majority of known oncogenic HPV types. Genomes from the same HPV type with 1-10% nucleotide differences designate HPV variant lineages. The established nucleotide variations observed in extant HPV genomes have been fixed through evolutionary processes prior to human population expansion and global dissemination. To characterize viral types and variants associated with pathology for clinical applications (e.g. screening), molecular epidemiological studies have proven essential for identifying links between HPV natural history and carcinogenicity. This chapter presents a historical account of HPV genomics in the context of major discoveries and advances over the past 2 thousand years.

Human papillomavirus genome variants.

Amongst the human papillomaviruses (HPVs), the genus Alphapapillomavirus contains HPV types that are uniquely pathogenic. They can be classified into species and types based on genetic distances between viral genomes. Current circulating infectious HPVs constitute a set of viral genomes that have evolved with the rapid expansion of the human population. Viral variants were initially identified through restriction enzyme polymorphisms and more recently through sequence determination of viral fragments. Using partial sequence information, the history of variants, and the association of HPV variants with disease will be discussed with the main focus on the recent utilization of full genome sequence information for variant analyses. The use of multiple sequence alignments of complete viral genomes and phylogenetic analyses have begun to define variant lineages and sublineages using empirically defined differences of 1.0-10.0% and 0.5-1.0%, respectively. These studies provide the basis to define the genetics of HPV pathogenesis.

Condylomatous genital lesions in cynomolgus macaques from Mauritius.

Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.

Human papillomavirus DNA methylation as a potential biomarker for cervical cancer.

Sexually transmitted carcinogenic human papillomavirus (HPV) infections are extraordinarily prevalent worldwide. However, most incident HPV infections clear within a few years, whereas a small minority persists to invasive cancer. Recent studies indicate that detection of methylated viral DNA may distinguish women with cervical intraepithelial neoplasia grade 2+ (CIN2+) from those with a carcinogenic HPV-type infection that shows no evidence of CIN2+. Several studies have reported a positive association between methylation of CpG sites in the L1 gene and CIN2+, although there are inconclusive results about methylation of CpG sites in the upstream regulatory region (URR). In this review, we summarize the current state of knowledge on HPV DNA methylation in cervical carcinogenesis, and discuss the merits of different methods used to measure HPV DNA methylation. To follow the promising leads, we suggest future studies to validate the use of methylated carcinogenic HPV DNA as a predictive and/or diagnostic biomarker for risk of cervical cancer among HPV-positive women.

The cervical microbiome over 7 years and a comparison of methodologies for its characterization.

BACKGROUND: The rapidly expanding field of microbiome studies offers investigators a large choice of methods for each step in the process of determining the microorganisms in a sample. The human cervicovaginal microbiome affects female reproductive health, susceptibility to and natural history of many sexually transmitted infections, including human papillomavirus (HPV). At present, long-term behavior of the cervical microbiome in early sexual life is poorly understood. METHODS: The V6 and V6-V9 regions of the 16S ribosomal RNA gene were amplified from DNA isolated from exfoliated cervical cells. Specimens from 10 women participating in the Natural History Study of HPV in Guanacaste, Costa Rica were sampled successively over a period of 5-7 years. We sequenced amplicons using 3 different platforms (Sanger, Roche 454, and Illumina HiSeq 2000) and analyzed sequences using pipelines based on 3 different classification algorithms (usearch, RDP Classifier, and pplacer). RESULTS: Usearch and pplacer provided consistent microbiome classifications for all sequencing methods, whereas RDP Classifier deviated significantly when characterizing Illumina reads. Comparing across sequencing platforms indicated 7%-41% of the reads were reclassified, while comparing across software pipelines reclassified up to 32% of the reads. Variability in classification was shown not to be due to a difference in read lengths. Six cervical microbiome community types were observed and are characterized by a predominance of either G. vaginalis or Lactobacillus spp. Over the 5-7 year period, subjects displayed fluctuation between community types. A PERMANOVA analysis on pairwise Kantorovich-Rubinstein distances between the microbiota of all samples yielded an F-test ratio of 2.86 (p<0.01), indicating a significant difference comparing within and between subjects' microbiota. CONCLUSIONS: Amplification and sequencing methods affected the characterization of the microbiome more than classification algorithms. Pplacer and usearch performed consistently with all sequencing methods. The analyses identified 6 community types consistent with those previously reported. The long-term behavior of the cervical microbiome indicated that fluctuations were subject dependent.

Classification and nomenclature system for human Alphapapillomavirus variants: general features, nucleotide landmarks and assignment of HPV6 and HPV11 isolates to variant lineages.

BACKGROUND: Papillomaviruses constitute a family of viruses that can be classified into genera, species and types based on their viral genome heterogeneity. Currently circulating infectious human Alphapapillomaviruses (alpha-PVs) constitute a set of viral genomes that have evolved from archaic times and display features of host co-speciation. Viral variants are more recently evolved genomes that require a standardized classification and nomenclature. OBJECTIVES: To describe a system for the classification and nomenclature of HPV viral variants and provide landmarks for the numbering of nucleotide positions. METHODS: The complete 8 kb genomes of the alpha-9 species group and HPV6 and 11 types, collected from isolates throughout the world were obtained from published reports and GenBank. Complete genomes for each HPV type were aligned using the E1 start codon and sequence divergence was calculated by global and pairwise alignments using the MUSCLE program. Phylogenetic trees were constructed from the aligned sequences using a maximum likelihood method (RAxML). RESULTS: Pairwise comparisons of nucleotide differences between complete genomes of each type from alpha-9 HPV isolates (HPV16, 31, 33, 35, 52, 58 and 67) revealed a trimodal distribution. Maximum heterogeneity for variants within a type varied from 0.6%-2.3%. Nucleotide differences of approximately 1.0%-10.0% and 0.5%-1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Analysis of 43 HPV6 complete genomes indicated the presence of 2 variant lineages, whereas 32 HPV11 isolates were highly similar and clustered into 2 sublineages. A table was constructed of the human alpha-PV landmark nucleotide sequences for future reference and alignments. CONCLUSIONS: A proposed nomenclature system for viral variants and coordination of nucleotide positions will facilitate the comparison of variants across geographic regions and amongst different populations. In addition, this system will facilitate study of pathogenic, tissue tropism and functional differences amongst variant lineages of and polymorphisms within HPV variants.

Moderate influence of human APOBEC3F on HIV-1 replication in primary lymphocytes.

Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.

The localization of APOBEC3H variants in HIV-1 virions determines their antiviral activity.

Several members of the human APOBEC3 family of cytidine deaminases can potently restrict retroviruses such as HIV-1. The single-domain APOBEC3H (A3H) is encoded by four haplotypes, of which only A3H haplotype II-RDD (hapII-RDD) restricts HIV-1 efficiently. The goal of this study was to elucidate the mechanisms underlying the differences in antiviral activity among A3H haplotypes. The naturally occurring A3H hapI-GKE and hapII-RDD variants differ at three amino acid positions. A panel of six site-directed mutants containing combinations of the three variable residues was used to determine A3H protein expression, requirements of A3H virion incorporation, and A3H-Gag interactions. The catalytic activity of each A3H protein was assessed directly by using an Escherichia coli mutator assay. We found that the incorporation efficiencies of A3H variants into HIV-1 virions were comparable despite major differences in cellular expression. An assessment of the enzymes' catalytic activities showed that the deaminase activity of each A3H variant correlated with protein expression, suggesting similar enzymatic efficiencies. Surprisingly, virion incorporation experiments using Gag deletion mutants demonstrated that A3H haplotypes interacted with different Gag regions. A3H hapII-RDD associated with nucleocapsid in an RNA-dependent manner, whereas A3H hapI-GKE associated with the C-terminal part of matrix and the N-terminal capsid domain. Our results show that the A3H hapII-RDD interaction with nucleocapsid is critical for its antiviral activity and that the inability of A3H hapI-GKE to interact with nucleocapsid underlies its limited antiviral potential. Thus, the antiviral activity of A3H haplotypes is determined by its incorporation into the viral core, in proximity to the reverse transcription complex.

Polymorphisms and splice variants influence the antiretroviral activity of human APOBEC3H.

Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity. Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.

Cytidine deamination induced HIV-1 drug resistance.

The HIV-1 Vif protein is essential for overcoming the antiviral activity of DNA-editing apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) cytidine deaminases. We show that naturally occurring HIV-1 Vif point mutants with suboptimal anti-APOBEC3G activity induce the appearance of proviruses with lamivudine (3TC) drug resistance-associated mutations before any drug exposure. These mutations, ensuing from cytidine deamination events, were detected in >40% of proviruses with partially defective Vif mutants. Transfer of drug resistance from hypermutated proviruses via recombination allowed for 3TC escape under culture conditions prohibitive for any WT viral growth. These results demonstrate that defective hypermutated genomes can shape the phenotype of the circulating viral population. Partially active Vif alleles resulting in incomplete neutralization of cytoplasmic APOBEC3 molecules are directly responsible for the generation of a highly diverse, yet G-to-A biased, proviral reservoir, which can be exploited by HIV-1 to generate viable and drug-resistant progenies.