scholarly journals Infectious Virions Produced from a Human Papillomavirus Type 18/16 Genomic DNA Chimera

2002 ◽  
Vol 76 (10) ◽  
pp. 4723-4733 ◽  
Author(s):  
Craig Meyers ◽  
Jennifer L. Bromberg-White ◽  
Jiaping Zhang ◽  
Michelle E. Kaupas ◽  
Janine T. Bryan ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Genomic Dna ◽  
Specific Antiserum ◽  
Structural Genes ◽  
Raft Culture ◽  
Dna Chimera ◽  
Nonstructural Genes

ABSTRACT The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.

scholarly journals In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1150 ◽  
Author(s):  
Vincenza De Gregorio ◽  
Francesco Urciuolo ◽  
Paolo Antonio Netti ◽  
Giorgia Imparato
Keyword(s):  
Human Papillomavirus ◽  
Tumor Microenvironment ◽  
Critical Role ◽  
Genetically Engineered ◽  
Cancer Disease ◽  
Raft Culture ◽  
Squamous Epithelia

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

scholarly journals Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

2016 ◽  
Vol 90 (10) ◽  
pp. 5047-5058 ◽  
Author(s):  
T. Klymenko ◽  
H. Hernandez-Lopez ◽  
A. I. MacDonald ◽  
J. M. Bodily ◽  
S. V. Graham
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Protein Expression ◽  
Capsid Protein ◽  
Drug Targets ◽  
Cell Metabolism ◽  
Dependent Manner ◽  
Control Activity

ABSTRACTThe human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytesin vitroandin vivothat HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression.IMPORTANCEHuman papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4^L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and the production of new virions.

scholarly journals CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function

2021 ◽  
Author(s):  
Apurva T. Prabhakar ◽  
Claire D. James ◽  
Dipon Das ◽  
Raymonde Otoa ◽  
Matthew Day ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Cellular Protein ◽  
Human Papillomaviruses ◽  
Retention Function ◽  
Critical Function ◽  
Human Papillomavirus 16 ◽  
Ck2 Inhibitors

AbstractDuring the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here, we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo, and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A activates and represses transcription identically to E2-WT (wild-type), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin when compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis when compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. We next tested whether this difference in E2-S23A levels during mitosis disrupts E2 plasmid retention function. We developed a novel plasmid retention assay and demonstrate that E2-S23A is deficient in plasmid retention when compared with E2-WT. siRNA targeted knockdown of TopBP1 abrogates E2-WT plasmid retention function. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, which is critical for E2 plasmid retention function and in HPV16 immortalization of keratinocytes.ImportanceHuman papillomaviruses are causative agents in around 5% of all cancers, with no specific anti-viral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex formation with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis and mediates plasmid retention by E2. This function promotes the partitioning of viral genomes into the nuclei of daughter cells following mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo, and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, demonstrating a critical function for this residue. Together, our results suggest that CK2 inhibitors may disrupt the E2-TopBP1 dependent HPV16 life cycle and potentially kill HPV16 positive cancers, which lays a molecular foundation to develop novel therapeutic approaches for combating HPV16 disease.

scholarly journals CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

mBio ◽  
2021 ◽  
Author(s):  
Apurva T. Prabhakar ◽  
Claire D. James ◽  
Dipon Das ◽  
Raymonde Otoa ◽  
Matthew Day ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Cellular Protein ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Human Papillomavirus 16 ◽  
Causative Agents ◽  
Mitotic Chromatin

Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo .

scholarly journals The Use of Chimeric Venezuelan Equine Encephalitis Viruses as an Approach for the Molecular Identification of Natural Virulence Determinants

2000 ◽  
Vol 74 (9) ◽  
pp. 4258-4263 ◽  
Author(s):  
Ann M. Powers ◽  
Aaron C. Brault ◽  
Richard M. Kinney ◽  
Scott C. Weaver
Keyword(s):  
Guinea Pigs ◽  
Size Analysis ◽  
Virulence Determinants ◽  
Plaque Size ◽  
Venezuelan Equine Encephalitis ◽  
Structural Genes ◽  
Nonstructural Genes ◽  
Chimeric Viruses

ABSTRACT Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.

scholarly journals ADAR1 function affects HPV replication and is associated to recurrent human papillomavirus-induced dysplasia in HIV coinfected individuals

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maria Pujantell ◽  
Roger Badia ◽  
Iván Galván-Femenía ◽  
Edurne Garcia-Vidal ◽  
Rafael de Cid ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Immune Activation ◽  
Hpv Infection ◽  
Innate Immune ◽  
Type I ◽  
Innate Immune Activation ◽  
Enhanced Expression

AbstractInfection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.

scholarly journals In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced.

1997 ◽  
Vol 41 (9) ◽  
pp. 1898-1903 ◽  
Author(s):  
D Sereno ◽  
J L Lemesre
Keyword(s):  
Life Cycle ◽  
Culture Conditions ◽  
Cross Resistance ◽  
Diminazene Aceturate ◽  
Drug Pressure ◽  
Mouse Macrophages ◽  
Wild Type Clone ◽  
Different Levels

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

scholarly journals Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

2020 ◽  
Vol 8 (5) ◽  
pp. 715 ◽  
Author(s):  
Samantha Gunasekera ◽  
Alireza Zahedi ◽  
Mark O’Dea ◽  
Brendon King ◽  
Paul Monis ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cells ◽  
Protozoan Parasites ◽  
Future Directions ◽  
In Vivo Models ◽  
Severe Diarrhea ◽  
Transformed Cell Lines ◽  
Gnotobiotic Piglets

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

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