HPV L1 Gene Expression In Yeast Cell-Free Lysate System And Primary Keratinocyte System

Papillomaviruses(PVs)are DNA viruses that infect keratinocytes in differentiating epithelia and induce a series of benign and malignant hyperproliferative lesions.To date, more than two hundreds of different HPV types have been identified.Infections by genital papillomaviruses are one of the most common sexually transmitted viral infections.The viruses that infect the genital tract are further classified as either high risk or low risk viruses.Low-risk HPV genotypes,such as HPV1,2,6,11,induce benign warts such as common warts and condyloma acuminata.In contrast,infections by high-risk HPV types including HPV 16,18,45,58 can lead to the development of cervical cancer which is one of the most common cancers worldwide.It is well known that HPVs are a family of approximately 8kb,non-enveloped and double-stranded DNA viruses.The genome of HPV can be divided into three regions:the non-coding region, the early and late regions.The late region includes two open reading frames(ORF),L1 and L2,which encode the major capsid protein and minor capsid protein assembling into the virus capsid together.However,the productive life cycle of HPVs is intimately linked to epithelial growth and cell differentiation.The expression of major capsid protein L1 only occurs in the terminally differentiated KCs.The link has posed a substantial barrier to the study of HPV in the laboratory because HPVs cannot be propagated in conventional cell lines.So far,different culture systems have been developed to mimick keratinocyte differentiation in vitro,however,it is unclear of the detailed mechanism responsible for the L1 gene expression,VLP assembly and DNA encapsidation.Further demonstration on these aspects will greatly help to the vaccine design,the anti-virus drug screen,and the research on the virus-cell interaction in the future.HPV 58 is an important HPV type tightly associated with cervical cancer.Several studies have suggested that HPV58 plays a prominent role in cervical cancer in Asia,and it is also the third most prevalent HPV type found in cervical cancer patients in China except HPV16 and 18.However,HPV58 has received little attention in the world.Considering the importance of HPV58 in China,studies on the HPV58 L1 protein expression and the development of its prophylactic vaccine is greatly desired.It has been demonstrated that the L1 sequences of the cervical carcinoma-associated HPV types always contain a second ATG codon downstream of the first translation initiation codon,which possess the ability to produce a variant L1 protein.The formation of virus-like particles(VLPs) occurs more efficiently when the second ATG codon is used for studies of the L1 gene expression in vitro.It has been postulated that the long L1 protein mainly occurs in the high-risk HPV-infected epithelium and plays a role in the carcinogenesis of cervical cancer.In contrast,the virus-like particles are mostly assembled by the short L1 protein. However,it is not clear whether the HPV 58 L1 gene is able to express two formats of the L1 proteins,with a long and a short L1 protein,which can self-assemble into virus-like particles.Also,little is known whether and how the HPV 58 L1 gene is expressed in different expression systems in which the L1 genes from the other HPV types are well expressed.In vitro translation system is a powerful tool to study gene expression and regulation. Yeast cell-free system(in vitro translation system)is a kind of in vitro translation system based on the extracts of the yeast cells.Yeast cell-free system has been widely used for the translation of various mammalian and viral mRNAs.Compared with other in vitro translation systems,yeast cell-free system shows a lot of advantages to study the regulatory mechanism of viral gene expression and its responses to the genetic and physiological changes.However,it is still unclear whether HPV mRNA could be translated in L1 protein in the yeast cell-free system.In this report,we first developed a new yeast cell-free lysate system that is suitable for efficient translation of HPV 58L1 mRNA.We successfully optimized the system in several aspects to achieve the optimal translation efficiency and demonstrated for the first time that HPV58 VLPs were assembled from the in vitro-translated L1 protein in yeast cell-free system.It is well known that complex mechanisms are involved in the regulation of PV late gene expression post-transcriptionally.However,no experimental data could clarify why the differentiated keratinocyte is permissive for the translation of the native L1 genes,and how the cell differentiation regulates the L1 gene expression freely.It has been proved that the codon usage of a target gene is tightly linked to its expression level in the host cells,and codon modification of PV early and late genes could greatly enhance their gene expression.It indicates that the match extent between codon usage and the tRNA species is an important determinant for viral gene expression,and the poor synthesis of PV L1 protein was due to the lack of specific tRNAs,especially some rare tRNAs. However,there are only very limited experimental data about the tRNA abundance and its regulatory function in gene expression.The detailed mechanism is still unclear.So efforts are still desirable to elucidate the mechanism of gene regulation in fine details.To explore the detailed molecular mechanism of L1 gene expression,we used the differentiating primary keratinocytes to establish the keratinocyte culture system and in vitro translation system,and investigated the transcription and translation pattern of both wild type and codon-modified HPV6b L1 genes during cell differentiation.Ⅰ.The in vitro translation of HPV58L1 mRNA in an optimized yeast cell-free systemWe first amplified the long and short L1 genes from the plasmid pLink322-HPV58 containing the complete genome of HPV58 using PCR method.The PCR products were then ligated into the eukaryotic expression vector pcDNA3.0 respectively to generate the constructs pcDNA3/LL1 and pcDNA3/SL1.The correct sequence and orientation of the L1 genes were further confirmed by the restriction analysis and DNA sequencing.Then the long and short L1 genes in the vector were in vitro transcribed into corresponding mRNAs using the in vitro transcription kit.To establish the yeast cell-free system,we used the protoplasts of the yeast cells to prepare the yeast cell-free lysate according to the previous procedure with some modification.Given that the concentration of the magnesium and potassium ions in the lysis buffer will greatly affect the translational activity of exogenous mRNAs,we optimized the concentration of them according to the requirements of the HPV58L1 mRNA translation.To test the effects of sucrose on the stability of the system,we applied different concentrations of sucrose during the lysate preparation.To determine the effects of optimization,we compared the translation efficacy between commercial rabbit reticulocyte system(RRL system)and the yeast cell-free system before and after optimization.The defined quantities of HPV6b L1 protein was used as a standard.Results showed that the in vitro translation system we established here is sufficient for the translation of HPV58L1 mRNA.The concentration of magnesium and potassium ions affects the L1 protein production greatly.The optimal concentration of magnesium was found to be 2.2mM,and that of potassium was 220mM.Intriguingly,sucrose seemed to provide a "cushion" that could assist the lysate against the environmentally thermometric changes and greatly increase the stability of the lysate.The optimal concentration of sucrose was 100mM.After optimization,the translation efficiency of HPV58L1 mRNA in the yeast cell-free system was twice higher than that of the RRL system.After calculation,50-70μg of the L1 protein expressed from 1μg of the short L1 mRNA in a volume of one ml translation reaction could be produced after optimization,which was significantly higher than that before optimization(20-40μg).To further explore the difference between HPV58 long and short L1 mRNA translation in the yeast in vitro translation system,we applied long and short L1 mRNAs into the system and performed the reaction by altering the reaction time,the amount of mRNA template,and by adding exogenous amino acids or leucine and yeast aminoacyl-tRNAs respectively.Results showed that both long and short L1 mRNAs could be translated in the yeast cell-free system.The translation could initiate within 10 min,but the level of the short L1 protein was significantly higher than that of the long L1 one.The short L1 protein level was slightly increased from 10 to 30 min in contrast with the long L1 protein level. The expression of both long and short L1 proteins was dramatically dependent on the amounts of the L1 mRNAs.Again,the level of the short L1 protein was significantly higher than that of the long L1 protein.Supplement of exogenous yeast aa-tRNAs slightly enhanced the translation of L1 mRNAs suggesting that endogenous aa-tRNAs were sufficient.Interestingly,supplement of the exogenous amino acids significantly enhanced translation of the long L1 mRNA,while hardly enhanced that of the short L1 indicating clearly that available endogenous amino acids are the major factor,which limits translation of the long L1 mRNA in the yeast cell-free lysates.To reveal the reason responsible for the difference between long and short L1 synthesis, exogenous leucine was supplied to the in vitro translation reaction.To clarify whether the in vitro translated L1 protein could self assemble into VLP particles,we applied the products to the sucrose ultracentrifugation and observed the formation of VLP directly. Western Blot analysis revealed that long L1 signal was greatly enhanced by the supplement of leucine in a dose-dependent manner.However,the short L1 signal had no response to it.So it seems that a single amino acid leucine can limit the rate of translation of the long L1 mRNA due to its high proportion in the L1 protein sequence. Electron microscopy result showed that both long and short L1 capsid proteins in the yeast lysate system could self-assemble into VLPs.Here we developed a yeast cell-free lysate system that was suitable for in vitro translation of HPV58L1 mRNA.The system was systematically optimized after which the translation efficiency was greatly enhanced and the VLP particles were observed.So the optimized yeast cell-free system was the second in vitro translation system sufficient for the HPV VLP formation except RRL system.Then we used this system to further explore the difference between long and short L1 mRNA translation and finally observed the VLPs assembled from both long and short L1 proteins.Considering that the HPV/yeast cell-free system is highly amendable and simple to be made,the system may be very useful in:1)exploring the factors affecting the expression of HPV58L1 protein in the yeast culture system,improving the protein production and modifying the vaccine design and development;2)clarifying the molecular mechanism of VLP assembly,increasing the VLP formation efficacy,and reducing the difference between the antigenicity of yeast-expressed VLP and native virions;3)revealing the molecular mechanism of DNA encapsidation,facilitating the research on the infectivity and infection mechanism of VLPs from both long and short L1 proteins,and providing possible new anti-virus targets.Currently,we have applied a national invention patent waiting for final approval.No similar reports have been found.Ⅱ.The HPV6b L1 gene expression in the primary keratinocyte systemTo explore the effects of codon composition on the L1 gene expression in the continuously differentiating keratinocytes(KCs),HPV6b wild type(Nat)and codon modified(Mod)L1 gene construct were transiently transfected into the human and mouse primary keratinocytes.Following transient transfection,cells were collected at D3,D6,D9,D12 post-transfection for RNA extraction,protein preparation and immunofluorescence staining.Quantitative RT-PCR was used to detect the L1 transcripts in the transfected KCs and Western Blot to determine the L1 protein synthesis.The morphological changes observed in the L1-transfected KCs over time reflected cell differentiation that was confirmed by the increased expression of involucrin,a marker of keratinocyte terminal differentiation,using immunofluorescence microscopy.QRT-PCR results showed that the transfected KCs could continuously transcribe the authentic and modified L1 mRNA for at least 12 days.The level of Mod L1 mRNA is higher than that of Nat L1 indicating that the codon modification promotes the transcription of L1 genes in KCs.The transcription of both L1 genes decreased as the cell differentiated.Western Blot analysis showed that the Nat L1 protein signal increased significantly over time.In contrast,Mod L1 signal decreased greatly over time.The results suggested that codon composition of the L1 mRNA plays a key role in regulation of the continuous expression of the L1 proteins.The expression of L1 protein was regulated in the post-transcription level and tightly associated with the cell differentiation.The result of the immunoprecipatation of[S35]methionine-labeled L1 protein showed that the detected continuous expression of the L1 protein was due to the continuous synthesis of the L1 protein and the codon composition of the L1 mRNA sequences was a major determinant of the continuous synthesis of the L1 protein.Considering that the natural host cell of HPV infection is human keratinocyte(HKC),we further explored the expression of L1 genes in HKC culture system.Similar results were obtained.To further confirm the regulatory role of tRNAs on the L1 gene translation,we extracted the aa-tRNAs from human and mouse KCs of different differentiation states and applied them to the KC in vitro translation system.Results showed that Nat L1 mRNA tended to be translated in the differentiated KCs,and the Mod L1 mRNA in the less-differentiated KCs.The result was consistent with those from the transfected KCs indicating that aa-tRNAs in less-differentiated and differentiated KCs differentially match the HPV Nat and Mod L1 mRNAs to regulate their translations in vitro.In this part,we first established the keratinocyte in vivo and in vitro systems using the continuously differentiating cells and investigated the regulatory mechanism of L1 gene expression.We showed for the first time the transcription and translation duration of L1 genes in the transiently transfected KCs,and revealed the tight link between isoencoding codons of viral gene and the host cell differentiation.It seems that the codon modification could change the duration and pattern of L1 gene expression in KCs which was mainly because the tRNA pools differ greatly during the cell differentiation,thus differentially regulate the transcription and translation of the Nat and Mod L1 mRNAs in vitro.Our results as important experimental data shed more light on the detailed regulatory mechanism of L1 gene expression.No similar reports were observed.