Human Papillomavirus 16 Variants from Zambian Women with Normal Pap Smears

Human papillomavirus (HPV) type 16 is the most prevalent high-risk viral genotype associated with cervical cancer. Six distinct phylogenetic clusters of HPVs have been identified and are distributed differently across five continents. HPV16 DNA was extracted from cervico-lavage samples from women with normal pap smears. The LCR regions were amplified in triplicate, cloned, sequenced, and analyzed from a total of 11 recovered HPV16 positive samples [Ng’andwe et al. (2007): BMC Infect Dis 7:77] were analyzed for sequence variation. The HPV16 LCR variants were assessed for promoter activity by use of a luciferase reporter gene. Six novel HPV16 variants with nucleotide exchanges in the LCR region were identified. Five clones were classified as European group HPV16 variants and one as an African group variant. Two of these variants had relatively lower promoter activity, 30% of that of the wildtype strain. The decreased promoter activity of some HPV16 variants may decrease expression of viral oncogenes and may be linked with the development, phenotype and severity of the cervical lesions in women infected with these across HPV16 variants

Human Papillomavirus 16 Variants from Zambian Women with Normal Pap Smears

Human papillomavirus (HPV) type 16 is the most prevalent high-risk viral genotype associated with cervical cancer. Six distinct phylogenetic clusters of HPVs have been identified and are distributed differently across five continents. HPV16 DNA was extracted from cervico-lavage samples from women with normal pap smears. The LCR regions were amplified in triplicate, cloned, sequenced, and analyzed from a total of 11 recovered HPV16 positive samples [Ng’andwe et al. (2007): BMC Infect Dis 7:77] were analyzed for sequence variation. The HPV16 LCR variants were assessed for promoter activity by use of a luciferase reporter gene. Six novel HPV16 variants with nucleotide exchanges in the LCR region were identified. Five clones were classified as European group HPV16 variants and one as an African group variant. Two of these variants had relatively lower promoter activity, 30% of that of the wildtype strain. The decreased promoter activity of some HPV16 variants may decrease expression of viral oncogenes and may be linked with the development, phenotype and severity of the cervical lesions in women infected with these across HPV16 variants

Viral trans-factor independent replication of human papillomavirus genomes

<p>Abstract</p> <p>Background</p> <p>Papillomaviruses (PVs) establish a persistent infection in the proliferating basal cells of the epithelium. The viral genome is replicated and maintained as a low-copy nuclear plasmid in basal keratinocytes. Bovine and human papillomaviruses (BPV and HPV) are known to utilize two viral proteins; E1, a DNA helicase, and E2, a transcription factor, which have been considered essential for viral DNA replication. However, growing evidence suggests that E1 and E2 are not entirely essential for stable replication of HPV.</p> <p>Results</p> <p>Here we report that multiple HPV16 mutants, lacking either or both E1 and E2 open reading frame (ORFs) and the long control region (LCR), still support extrachromosomal replication. Our data clearly indicate that HPV16 has a mode of replication, independent of viral trans-factors, E1 and E2, which is achieved by origin activity located outside of the LCR.</p

DNA Replication and Genome Maintenance of Human Papillomavirus Type 16 in Mammalian Cells

Human papillomavirus (HPV) is a major causative agent of cervical cencer and a number of human cancers. The high‐risk types can be detected in more than 90% of cervical cancer, of which HPV16 is the most common found. The virus establishes a latent infection in basal epithelial cells in which the viral genomes can be stably maintained as extrachromosomal DNA for decades before the development of cancer. Several attempts have been made in bovine papillomavirus (BPV) to understand how the virus can maintain its genome at constant copy numbers in dividing cells. The viral protein E2 has been proposed to serve as a molecular linker that tethers the viral genome to host chromosomes. However, there is a great deal of dispartities between BPV and HPV with respect to variations of E2 binding pattern, cellular targets, and number of E2 binding sites within their genomes. It is unclear whether HPV utilizes the same maintenance mechanism. Papillomaviruses utilize two viral factors, E1, a DNA helicase, and E2, a transcriptional activator and auxiliary replication factor for replication and DNA maintenance in host cells. However, previous studies in yeast and findings reported here demonstrated that HPV16 can replicate independently of the viral E1 and E2 proteins. It was also shown that HPV16 possibly contains alternate origin of replication outside the LCR region that relies entirely on cellular replication proteins. In this study, we further identified cis‐elements and trans‐acting factors that are required for HPV genome maintenance during persistent infection. Two distinct regions have been mapped as maintenance elements as they provided DNA stability in mammalian cells in the absence of any viral proteins. We found three nonamer sites (TTAGGGTTA) which resemble telomeric repeats on the cis‐maintenance elements within the late region and observed altered expression of proteins associated with telomeric repeats in HPV16 immortalized cells. The telomere binding proteins such as telomeric repeat binding factors 1(TRF1),TRF2, TRF2‐interacting protein hRAP, the telomere‐associated poly (ADP‐ribose) polymerase (tankyrase) were previously found on EBV latent origin of replication and contributed to episomal maintenance. These observations motivated us to hypothesize that HPV16 has evolved similar mechanism to maintain its genome in host cells. Using ChIP assay, we found that TRF2, protection of telomere 1 (POT1) and a RecQ helicase WRN bound to the putative binding sites within HPV16 genome. Deletion mutations of TRF binding sites altered the plasmid maintenance activity suggesting the implication of these binding sites as well as the neighboring sequences in HPV life cycle. These results imply that the telomere binding factors are novel cellular factors for HPV16 DNA maintenance. Furthermore, several binding sites for topoisomeraseII (TopoII), centromere binding protein CENP‐B, and high mobility group HMG were also predicted in the late region suggesting that HPV maintenance is regulated by multiple cellular factors

Analysis of \u3ci\u3ecis\u3c/i\u3e-elements that facilitate extrachromosomal persistence of human papillomavirus genomes

Human papillomaviruses (HPVs) are maintained latently in dividing epithelial cells as nuclear plasmids. Two virally encoded proteins, E1, a helicase, and E2, a transcription factor, are important players in replication and stable plasmid maintenance in host cells. Recent experiments in yeast have demonstrated that viral genomes retain replication and maintenance function independently of E1 and E2 [Angeletti, P.C., Kim, K., Fernandes, F.J., and Lambert, P.F. (2002). Stable replication of papillomavirus genomes in Saccharomyces cerevisiae. J. Virol. 76(7), 3350-8; Kim, K., Angeletti, P.C., Hassebroek, E.C., and Lambert, P.F. (2005). Identification of cis-acting elements that mediate the replication and maintenance of human papillomavirus type 16 genomes in Saccharomyces cerevisiae. J. Virol. 79(10), 5933-42]. Flow cytometry studies of EGFP-reporter vectors containing subgenomic HPV fragments with or without a human ARS (hARS), revealed that six fragments located in E6–E7, E1–E2, L1, and L2 regions showed a capacity for plasmid stabilization in the absence of E1 and E2 proteins. Interestingly, four fragments within E7, the 3′ end of L2, and the 5′ end of L1 exhibited stability in plasmids that lacked an hARS, indicating that they possess both replication and maintenance functions. Two fragments lying in E1–E2 and the 3′ region of L1 were stable only in the presence of hARS, that they contained only maintenance function. Mutational analyses of HPV16-GFP reporter constructs provided evidence that genomes lacking E1 and E2 could replicate to an extent similar to wild type HPV16. Together these results support the concept that cellular factors influence HPV replication and maintenance, independently, and perhaps in conjunction with E1 and E2, suggesting a role in the persistent phase of the viral lifecycle

Viral \u3ci\u3etrans\u3c/i\u3e-Factor Independent Replication of Human Papillomavirus Genomes

Background: Papillomaviruses (PVs) establish a persistent infection in the proliferating basal cells of the epithelium. The viral genome is replicated and maintained as a low-copy nuclear plasmid in basal keratinocytes. Bovine and human papillomaviruses (BPV and HPV) are known to utilize two viral proteins; E1, a DNA helicase, and E2, a transcription factor, which have been considered essential for viral DNA replication. However, growing evidence suggests that E1 and E2 are not entirely essential for stable replication of HPV. Results: Here we report that multiple HPV16 mutants, lacking either or both E1 and E2 open reading frame (ORFs) and the long control region (LCR), still support extrachromosomal replication. Our data clearly indicate that HPV16 has a mode of replication, independent of viral trans-factors, E1 and E2, which is achieved by origin activity located outside of the LCR

A Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Interacts with Notch1 and Notch3▿

Like other Alphaherpesvirinae subfamily members, bovine herpesvirus 1 (BHV-1) establishes latency in sensory neurons. The latency-related RNA (LR-RNA) is abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 is not the only viral product expressed during latency, but it is important for the latency reactivation cycle because it inhibits apoptosis. In this study, a yeast 2-hybrid screen revealed that ORF2 interacted with two cellular transcription factors, Notch1 and Notch3. These interactions were confirmed in mouse neuroblastoma cells by confocal microscopy and in an in vitro “pulldown” assay. During reactivation from latency, Notch3 RNA levels in trigeminal ganglia were higher than those during latency, suggesting that Notch family members promote reactivation from latency or that reactivation promotes Notch expression. A plasmid expressing the Notch1 intercellular domain (ICD) stimulated productive infection and promoters that encode the viral transcription factor bICP0. The Notch3 ICD did not stimulate productive infection as efficiently as the Notch1 ICD and had no effect on bICP0 promoter activity. Plasmids expressing the Notch1 ICD or the Notch3 ICD trans-activated a late promoter encoding glycoprotein C. ORF2 reduced the trans-activation potential of Notch1 and Notch3, suggesting that ORF2 interfered with the trans-activation potential of Notch. These studies provide evidence that ORF2, in addition to inhibiting apoptosis, has the potential to promote establishment and maintenance of latency by sequestering cellular transcription factors