Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

Kang, Bum Yong; You, Hong; Bandyopadhyay, Sarmistha; Agrawal, Nalini; Melchert, Russell B.; Basnakian, Alexei G.; Liu, Yong; Hermonat, Paul L.

doi:10.1186/1471-2180-9-79

Cited by 12 publications

(11 citation statements)

References 44 publications

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“…Additionally AAV2 is known to autonomously replicate in differentiating skin cells [12]–[15]. We therefore utilized the organotypic epithelial raft culture system (skin raft) to analyze effects of X on AAV2 autonomous DNA replication.…”

Section: Resultsmentioning

confidence: 99%

The X Gene of Adeno-Associated Virus 2 (AAV2) Is Involved in Viral DNA Replication

2014

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Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3′ end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3′ end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5′ AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

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Section: Resultsmentioning

confidence: 99%

The X Gene of Adeno-Associated Virus 2 (AAV2) Is Involved in Viral DNA Replication

2014

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“…Comparing three primary isolates and two established cervical cancer cell lines to normal keratinocytes Kang et al could detect overexpression of RPA, RFC, PCNA and DNA polymerase, which seem to play a role in adeno-associated virus DNA replication. 63 Increased expression of sine oculis homebox homologue 1 (SIX1, a master regulator of DNA replication in cervical cancer cells) can be induced by the E7 oncoprotein of HPVs in cervical intraepithelial neoplasia and cervical cancer and can result in higher levels of expression of genes related to the initiation of DNA replication such as RFC4. 69 Recently Niu et al showed that dysregulation of CDKN2A, IL1R2 and RFC4 may contribute to cervical cancer progression and may be potential diagnostic markers.…”

Section: Discussionmentioning

confidence: 99%

Potential new biomarkers for squamous carcinoma of the uterine cervix

et al. 2018

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AimAn in silico pathway analysis was performed in an attempt to identify new biomarkers for cervical carcinoma.MethodsThree publicly available Affymetrix gene expression data sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total 9 cervical cancer cell lines, 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples. An Agilent data set (GSE7410; 5 normal cervical samples, 35 samples from invasive cervical cancer) was selected as a validation set. Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. We compared the lists of differentially expressed genes between normal and CIN3 samples on the one hand (n=1923) and between CIN3 and invasive cancer samples on the other hand (n=628).ResultsSeven probe sets were identified that were significantly overexpressed (at least 2 fold increase expression level, and false discovery rate <5%) in both CIN3 samples respective to normal samples and in cancer samples respective to CIN3 samples. From these, five probes sets could be validated in the Agilent data set (P<0.001) comparing the normal with the invasive cancer samples, corresponding to the genes DTL, HMGB3, KIF2C, NEK2 and RFC4. These genes were additionally overexpressed in cervical cancer cell lines respective to the cancer samples. The literature on these markers was reviewedConclusionNovel biomarkers in combination with primary human papilloma virus (HPV) testing may allow complete cervical screening by objective, non-morphological molecular methods, which may be particularly important in developing countries

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“…Low expression of DNA polymerase δ-associated factors could explain the reduced conversion of incoming virus DNA in bovine cells, as these cellular components are known participants in initial replication of parvovirus genomes. Increased expression of proliferating cell nuclear antigen in certain cervical cancer isolates has been shown to enhance DNA replication of the parvovirus adeno-associated virus (Kang et al , 2009), suggesting that the expression level of these factors can alter parvovirus permissivity.…”

Section: Discussionmentioning

confidence: 99%

Differential replication of two porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression

Fernandes

Boisvert

Szelei

et al. 2014

Journal of General Virology

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Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5 kb genome expresses three major non-structural proteins (NS1, NS2 and SAT), and two structural proteins (VP1 and VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines have been shown to replicate the virus efficiently. Cell lines were established from a primary culture of bovine cells that allowed allotropic variants of PPV to be distinguished. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared with porcine cells, while production of infectious virus of the Kresse strain was barely detectable. Restriction points in these cells were the initial generation of DNA replication intermediates and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant were also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains, although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlight the dependent nature of parvovirus tropism on host factors and suggest that cells from a non-host origin can fully support a productive infection by both strains.

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Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

Cited by 12 publications

References 44 publications

The X Gene of Adeno-Associated Virus 2 (AAV2) Is Involved in Viral DNA Replication

The X Gene of Adeno-Associated Virus 2 (AAV2) Is Involved in Viral DNA Replication

Potential new biomarkers for squamous carcinoma of the uterine cervix

Differential replication of two porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression

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