Differential expression of papillomavirus L1 proteins encoded by authentic and codon modified L1 genes in methylcellulose-treated mouse keratinocytes
© Wang et al; licensee BioMed Central Ltd. 2007
Received: 05 October 2007
Accepted: 25 November 2007
Published: 25 November 2007
Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions. Here, we used methylcellulose to induce cell differentiation of primary mouse keratinocytes (KCs) in in vitro culture and assessed the expression of authentic and codon-modified version of L1 capsid genes from two PV types (HPV6b and BPV1). Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs. Western blot showed that methylcellulose significantly increased the levels of the L1 proteins expressed from two authentic L1 genes. Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs. These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.
Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions . Amplification of PV DNA and transcription of PV late genes is activated in suprabasal cells of differentiated epithelium, indicating that the PV life cycle is closely linked to host cell differentiation . This link has posed a substantial barrier to the study of PV in the laboratory because PVs cannot be propagated in conventional cell lines. Different raft culture systems that mimick keratinocyte differentiation in vitro have been developed to study viral gene transcription  and to achieve differentiation-specific viral amplification and virion morphogenic stages  and to produce virions from infected cells for sexually transmitted HPV types [5, 6]. However, the yield of infectious virus is very low in those systems. Because raft culture is a time-consuming technique, it cannot be used for rapid analysis of multiple constructs .
Recently, we established mouse primary KCs culture system to express PV L1 proteins by transient transfection of authentic or codon modified L1 gene expression constructs . Using the KC culture system, we proved that KC differentiation differentially regulates expression of PV authentic and codon modified L1 genes [8, 9]. Methylcellulose is a cell differentiation enhancer widely used in the study of KC differentiation [10–12]. Human KCs grown in methylcellulose semisolid medium for 48 h were induced to differentiate and express involucrin a terminal KC differentiation marker [13, 14]. In HPVs, Flores and Lambert reported that HPV 16 DNA replication was promoted and virus-like particles were detected when HPV-16-positive cervical epithelial cells were grown in medium containing 1.68% methylcellulose for 2 to 10 days . Methylcellulose also induced HPV31-positive epithelial cells to express two KC terminal differentiation markers involucrin and transglutaminase . However, no HPV 31 L1 protein expression was detected in HPV-infected KCs treated by methylcellulose although L1 mRNA was well transcribed . In this work, we investigated effects of methylcellulose treatment on expression of PV L1 genes in our established mouse primary KC culture system. Four PV L1 gene expression constructs including two authentic (Nat) L1 gene plasmids (pcDNA3HPV6b Nat L1 and pcDNA3BPV1 Nat L1) and two codon modified (Mod) L1 gene plasmids (pcDNA3HPV6b Mod L1, and pcDNA3BPV1 Mod L1) were used in the experiments as previously described .
The close association of the HPV life cycle with the differentiation state of its host cell is demonstrated by the restriction of late gene transcription and amplification of viral DNA to suprabasal epithelial cells. The study of HPVs in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Although raft culture system can provide a spatial separation of cells for the study of HPV life cycle , it is technically challenging and requires extended periods of time for KC growth and differentiation. Meantime, it is hard to isolate separate layers in raft culture system. We developed the simple mouse primary KCs culture system to successfully express PV L1 proteins by transient transfection of the L1 expression constructs . We reported that primary KCs in culture undergo cell differentiation to regulate expression of the PV L1 genes. Here, we demonstrated that suspension of mouse primary KCs in methylcellulose resulted in the rapid cell differentiation. As a model inducer of KC differentiation, use of methylcellulose has allowed us to characterize expression of targeted genes including L1 and involucrin in only 2–3 days instead of the 2 weeks required for raft culture and to study the mechanisms which regulate differentiation-dependent expression of the PV late genes. Methylcellulose did not influence L1 mRNA transcription in L1-transfected KCs, thus, the results confirmed that the expression of the L1 protein was post-transcriptionally regulated, consistent with previous studies [4, 17]. Our results demonstrated further that the L1 gene codon composition correlated with the differentiation-dependent expression of the L1 protein in L1-transfected KCs grown in KC-SF complete medium with or without methylcellulose. This correlation can be well explained by our previous observations that composition of aminoacyl-tRNA pool changes during cell differentiation, which differentially favors translation of PV authentic and codon-modified L1 genes .
In conclusion, we established a methylcellulose culture system in the mouse primary KCs and demonstrated that methylcellulose enhanced KC differentiation. Methylcellulose did not influence L1 transcription but differentially regulated translation of the authentic and codon-modified L1 genes. These data support our previous study that L1 expression in response to differentiation is regulated at the post-transcriptional level.
This work was funded in part by a National Health and Medical Research Council of Australia Industry Research Fellowship (301256 to KNZ) and the Queensland Cancer Fund (401623 to KNZ).
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