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ISSN 0582-9879                                    ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(1): 27-34                                    CN 31-1300/Q

Expression of Human Papillomavirus Type 6 L1 and L2 Isolated in China and Self Assembly of Virus-like Particles by the Products

WANG Miao, WANG Li-Liang, CHEN Lian-Feng, HAN Ye-Hua, ZOU Yu-Hong, SI Jing-Yi, SONG Guo-Xing*

( Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China )

 

Abstract      To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of≈4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 – 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.

 

Key words     HPV-6; L1; L2; baculovirus vector; virus-like particle

 

Human papillomaviruses (HPVs) infect cutaneous, genital, and respiratory epithelia in a tissue-specific manner. Infection with HPVs is widespread in the general population, and viral infection is closely associated with both benign and malignant lesions. From over 100 HPV types described[1], HPV-16 and 18 are strongly associated with high-grade anogenital lesions and invasive cancers[2]. HPV-6 and 11, only rarely associated with malignancies, are causative agents for about 90% of condyloma acuminata, benign lesions of the genital mucosa, with HPV-6 being detected more often in these lesions than HPV-11[3].

Papillomaviruses are non-enveloped viruses with a double-stranded DNA genome of 7.8 to 8 kb. Their icosahedral capsid is composed of the major capsid protein L1 and the minor capsid protein L2. It has been shown that for bovine papillomavirus type 1 (BPV-1) and HPV-1, 72 capsomers, each presumable a pentamer of L1, are arranged in a T=7 icosahedral lattice to form the virion capsid[4]. This structure seems to be highly conserved among papillomaviruses. The exact position of L2 within the capsid and the capsomer is still unknown. Since HPV virions cannot be propagated easily in vitro, nor obtained in reasonable quantities from lesions in vivo, it is rather difficult to study the properties of the capsid proteins and the mechanisms of their assembly into capsids.

Recombinant DNA techniques have been developed to assemble synthetic papillomavirus virions. Using vaccinia virus expression vectors recombined with HPV-16 L1 and L2 genes, Zhou et al.[5] detected approximately 40 nm particles which lacked the characteristic morphology of naturally occurring HPV-1 and BPV-1 particles. Using the more efficient baculovirus system, Kirnbauer et al.[6] obtained HPV-16 VLPs self assembled by the L1 or L1+L2 proteins, Rose et al.[7] obtained HPV-11 VLPs by expression of L1 in Sf9 cells. As for HPV-6, L1 and L1+L2 VLPs were obtained by expression in yeast cells[8,9], and L1 VLPs purified from baculovirus infected Sf9 cells were used for seroprevalence test[10]. HPVs isolated from different regions have variations in L1 ORF, which may affect assembling ability of the translated products into VLPs. In the present study we reported HPV-6 late region sequences (including L1 and L2 coding sequences) cloned from Chinese patients with condyloma acuminatum, demonstrated efficient production of HPV-6 VLPs by expression of L1 or L1 plus L2 in Sf9 cells, and probed the effects of L2 on L1 expression and on VLP assembly.

1    Materials and Methods

1.1   Cloning of HPV-6 L1 and L2 genes

HPV-6 DNA was isolated from genital wart biopsies (sample J was from Jinzhou, China and sample X from Xinxiang, China) as previously described[11], and used as amplifying template. Fragment DNA encoding L1 and L2 protein of HPV-6 were generated by PCR with Taq DNA polymerase (Promega). The sense (P1:5'-CCGGATCCBamHI-AATA5789TGTGGCGGCCTAGCGACAGCA-3') and anti-sense (P2:5'-CAGGATCCBamHIT7291TACCTTTTAGT-TTTGGCGCGCTT-3') primer set specific to L1 gene of HPV-6, and the sense (P3:5'-GCAGATCTBglIIAATA4423TGGCACATAGTAGGGCCCGACGACG-3') and antisense (P4:5'-CTAGATCTBglIIC5802TAGGCCGCCACATCTGAAAAAAATAAGGG-3')primer set specific to L2 gene of HPV-6 were synthesized according to the prototype HPV-6b sequence[12], and used in the PCR. BamHI and BglII sites added in the primers are indicated in bold and lower footnote number of a base shows its position in the prototype genome sequence.

Following gel purification, the PCR products were cloned into the pGEM-T vector (Promega) according to the manufacturer’s instructions. The inserted L1 and L2 fragments were sequenced three times (with Big-Dye DNA sequencing reaction kit from PE and ABI 310 sequencing instrument). Based upon determined upstream sequences internal sequencing primers (5'-C6205AGGGTTAATGTAGGTATGGA6225-3' for L1; 5'-A4816CATCCTCTGAAACAACTACCC4837-3' for L2) were synthesized and used in the sequencing.

1.2   Construction of recombinant baculovirus vectors

Bac-to-Bac baculovirus expression systems (Gibco BRL) was used for gene expression and detailed methods for baculovirus manipulation were referred to the instruction manual. In brief, the HPV-6 L1 and L2 genes from clinical sample J were excised from the recombinant pGEM plasmids by BamHI and BglII digestion respectively, gel purified, and separately subcloned into the BamHI-cut, dephosphorylated baculovirus donor plasmid pFastBac I downstream of the polyhedrin promoter. After characterization by restriction digestion and DNA sequencing, each of the recombinant plasmids was used to transform DH10Bac. Through Tn7 transposon-mediated site-specific in vivo transposition foreign gene expression cassette was integrated into a baculovirus shuttle vector (bacmid). The recombinant baculovirus DNA was isolated and used for transfection of Sf9 cells. Recombinant baculoviruses were harvested thereafter and were purified by plaque screening. The correctness of recombination was verified by PCR with L1 and L2 gene specific primers and the suggested sequencing primers (M13/pUC forward and reverse primers) provided in the bacmid DNA.

1.3   L1 and L2 protein expression assay

Sf9 cells were incubated at 28 ℃ in TC-100 medium supplemented with 10% fetal bovine serum. For the expression assay, 3×105 Sf9 cells in 0.5 ml fresh medium were infected with a baculovirus at a multiplicity of infection (MOI) of 10 in a well of 24-well plate. In the case of coinfection with L1 and L2 recombinant baculoviruses, total MOI was adjusted to 10 (i.e. MOI of L1-recombinant baculovirus + MOI of L2-recombinant baculovirus=10). 72 h post-infection (72 hpi), medium was discarded, and cells were washed twice with phosphate-buffered saline (PBS). After removing the supernatant thoroughly, 1×SDS-PAGE sample buffer(150 μl per well) was added to each well, the lysates of each well were transferred to 100 ℃ water bath for 5 min, the denatured samples were clarified by centrifugation at 8000 r/min for 2 min. Electrophoresis of 20 μl per sample in discontinuous SDS-PAGE (5％ stacking gel, pH 6.8; 10％ separate gel, pH 8.8) was followed by Coomassie blue R250 staining or Western blot (ECL Western blot fluorescent detection kit: Amersham Pharmacia). The stained gels were scanned and quantitatively analyzed with TotalLab software for protein molecular weight and composition percentage. Protocol for ECL Western blot fluorescent detection was carried out according to manufacturer suggestion.

1.4   Antibodies used in immuno-detection

Mouse anti-HPV-6 L1 VLP monoclonal antibodies H6C6, H6E51 and H6K57 were kindly provided by Christensen Milton S. Hershey Medical Center; Rabbit anti-HPV-6 L1-VLP and L2 polyclonal antibodies were obtained as a gift from Ian H. Frazer at University of Queensland, Australia and Denise A Galloway at Fred Hutchinson Cancer Research Center, University of Washington, respectively; Horseradish peroxidase (HRP) conjugated goat anti mouse/rabbit IgG antibodies and gold-conjugated (10 nm particles) goat anti rabbit IgG antibody were purchased from Beijing Zhongshan Biotechnology Co. Ltd.

1.5   Electron microscopy

A flask (25 cm2) of infected cells (MOI=10, 72 hpi) were harvested, washed with PBS and centrifuged at 1000 r/min for 5 min. The cell pellets were fixed in 3.8% glutaraldehyde in PBS for 24 h at 4 ℃. After fixation, the cells were washed three times in PBS (8 h each time) at 4 ℃ and post-fixed with 1% osmium tetroxide for 2 h at 4 ℃. The samples were then dehydrated with acetone of a series of graded concentration, and embedded in 1 ∶1 acetone-Epon 812 for 30 min at room temperature, further embedded in Epon 812 overnight at 37 ℃, and then polymerized for 24 h at 60 ℃. Ultrathin sections were cut with LKBIII ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, washed, dried and finally examined under JEM 1010 transmission electron microscope (TEM).

1.6   Production of virus-like particles (VLPs)

3×107 Sf9 cells were grown at 28 ℃ as adherent culture in a 175 cm2 flask containing 35 ml TC-100 insect medium with 10% fetal bovine serum, 100 u/ml penicillin, 100 mg/L streptomycin and 0.25 mg/L amphotericin B. For production of VLPs, 10 flasks of above cell cultures were infected with L1 recombinant baculovirus at a MOI of 10 or co-infected with L1 plus L2 recombinant baculoviruses at a MOI of 10 for each baculovirus. After 72 h, the cells were collected, centrifuged at 1000 g for 5 min and washed twice with PBS, and the final pellet was either stored in liquid nitrogen or processed immediately. The cell pellet was re-suspended in 10 ml PBS and purification procedure was essentially as described elsewhere[6], except that 325 g/L sucrose cushion in PBS and 307 g/L CsCl in PBS were used in ultracentrifugation of VLPs.

1.7   Electron microscopy and immunoelectron microscopy of VLPs

Samples to be assayed for the presence of VLPs were spun onto carbon-coated 300 mesh copper grids for 30 s, liquid was absorbed off with a paper towel. Negative staining was performed using routine method. The samples were stained with 2% phosphotungstic acid for 30 s, dried under light, and were observed under JEM 1010 TEM.

For immunoelectron microscopy, particles were absorbed onto carbon-coated nickel grids for 10 min in a box with a wet towel in it, and, after removing liquid, blocked in blocking solution (1%BSA-PBS) for 10 min, then incubated with a 1/5 dilution of first antibody in blocking solution at room temperature for 2 h. Rabbit polyclonal antibodies to HPV-6 L1 or HPV-6 L2 were used. Samples were then washed with blocking solution, and further incubated at room temperature for 1 h in a 1/25 dilution of gold-conjugated goat anti-rabbit IgG antibody in blocking solution. After sequential washing with blocking solution and water, the samples were negatively stained as above.

1.8   VLP ELISA

Purified VLP (2 mg/L per well in PBS) were added to wells of Nunc MaxiSorp plates and incubated at 37 ℃ for 1 h, then at 4 ℃ overnight. Following this incubation, plates were rinsed three times with PBS and then incubated for 1 h at room temperature with 50 μl of blocking agent (5% instant nonfat dry milk in PBS). Plates were again rinsed three times with PBS, 50 μl of diluted first antibodies(in 1% instant nonfat dry milk in PBS) was added to each well, and then plates were incubated at room temperature for 2 h. Naive mouse serum was used as negative control. The plates were rinsed five times with PBS, and 50 μl of a HRP-conjugated secondary antibody (1/1000 diluted in 1% instant nonfat dry milk in PBS) was added to each well and then incubated for 1 h at 37 ℃. The plates were rinsed 7 times with PBS, and 50 μl of peroxidase substrate buffer [2.43 ml 0.1 mol/L citric acid+2.57 ml 0.2 mol/L Na2HPO4+5 ml H2O+4 mg ortho-phenylenediamine (OPD)+15 μl 30% H2O2] was added and incubated for 5-10 min at 37 ℃. The reaction was terminated by the addition of 50 μl of 2 mol/L H2SO4. The A was measured in a microplate reader at 490 nm.

2    Results

2.1   Sequences of the cloned L1 and L2

The coding sequences of HPV-6 L1 or L2 were separately PCR amplified from clinical biopsies, cloned and sequenced. Basing upon overlapping sequence, L1 and L2 sequences from the same biopsy were assembled into one sequence of HPV-6 genome late region. Two such sequences (designated as JL2+1 and XL2+1, GenBank accession number AY015006 and AY015008) were obtained. Phylogenetic analysis (Fig.1) was performed between the two sequences and the published corresponding sequences of HPV-6a[8] and the prototype HPV-6b[12] with DNASTAR software (Jotun Hein Method). The cloned sequences were classified into HPV-6b. Compared with the prototype HPV-6b sequence, overall nucleotide variation in the late region sequences was less than 0.28%, and variation in L1 ORF and L2 ORF was less than 0.27% and 0.37%, respectively. The variations are summarized in Table 1.

 

Fig.1       Phylogenetic analysis (by Jotun Hein Method) of the clinical isolates of HPV-6 genome late region (JL2+1 and XL2+1) with prototype HPV-6b (L2+1-6b) and HPV-6a (L2+1-6a)

 

Table 1   Summary of variations in the cloned HPV-6 genome late region

 

JL2+1

XL2+1

L2 coding sequence

(4423→5802 in reference sequence) 1380 bp long, encodes 459 amino acids

 

A1814603CG→GCG(Thr61→Ala)

TCT3094731→TCC(Ser103→Ser)

 

G7965218AT→AAT(Asp266→Asn)

CAG9845406→CAA(Gln328→Gln)

CT11875609G→CCG(Leu396→Pro)

 

L1 coding sequence

(5789→7291 in reference sequence) 1503 bp long, encodes 500 amino acids

 

TA2005988C→TGC(Tyr67→Cys)

ACA8106598→ACT(Thr270→Thr)

GAA12937081→GAG(Glu431→Glu)

AAG13117099→AAA(Lys437→Lys)

Lower footnote number of base shows position in prototype genome sequence[12] while upper footnote number indicates position relative to the first nucleotide in the corresponding ORF, and upper footnote number of amino acid shows position in the translated protein sequence.

 

2.2   L1 and L2 protein expression in insect cells

The L1 and L2 coding sequences of XL2+1 were separately used to generate recombinant baculoviruses, yielding Bac6L1 and Bac6L2, respectively. SDS-PAGE analysis of total proteins from Sf-9 cells infected with Bac6L1 demonstrated a novel 54.5 kD protein seen by Coomassie blue staining [Fig. 2 (A2)]. This protein was not present in BacNR (non-recombinant baculovirus) infected cell lysate and comigrated with a protein that was immunoreactive with H6C6 monoclonal antibody prepared against HPV-6 L1 [Fig.2(A2')]. Compared with BacNR, Bac6L2 infected cell lysate demonstrated a novel 72.0 kD protein in SDS-PAGE [Fig.2(B2)], which was immuno-reactive with rabbit polyclonal antibody against HPV-6 L2 [Fig.2(B2')]. Lower and higher molecular weight L2-immunoreactive bands were also detected but not detected in control. L1 and L2 products constituted 6.82% and 5.03% of total cell proteins, respectively. The expressed L1 and L2 was localized in cell nucleus after synthesis in cytoplasm(immuno-histochemistry and laser confocal data not shown).

 

Fig.2       Expression of L1 and L2 in Sf9 cells and the Western blot analysis

M, protein marker with relative molecular weight indicated; A1, B1, Sf9 cell infected with BacNR (non-recombinant baculovirus) as control; A2, Sf9 cell infected with Bac6L1; B2, Sf9 cell infected with Bac6L2; A1', A2', B1', B2' are Western blot of A1, A2, B1, B2, respectively. Arrows indicate the bands of L1 and L2.

 

2.3   Electron microscopy of infected Sf9 cells

Electron micrograph of thin section of Bac6L1 infected Sf9 cells showed distinct VLPs (HPV capsid-like particles) in the nuclei of the cells [Fig.3(B)]. No such structures were seen in BacNR and Bac6L2 infected cells [Fig.3(A),(C)]. Baculoviruses were seen in all infected cell nuclei [Fig.3(A),(B),(C)].

 

Fig.3       Electron micrograph of baculovirus-infected Sf9 cells

Note that large amount of VLPs indicated by upward arrows were assembled in Sf9 cells (nuclei) infected with Bac6L1(B), but not in those infected with BacNR (A: negative control) or with Bac6L2(C). Baculoviruses indicated by rightward arrows (for longitudinal section) and leftward arrows (for cross section) were found in all infected Sf9 cells(A, B and C). The VL structure was distinguished from baculovirus seen in cross section in that the VLPs were not surrounded by a membrane-like structure while baculoviruses were often multiply occluded in a membrane-like structure, and individual baculovirus (whether or not stained centrally) had a clearer boundary than VLP.

 

2.4   Self-assembly of VLPs by L1 alone or by L1 plus L2

VLPs were purified from large-scale cell cultures. The band corresponding to VLPs in cesium chloride gradient ultracentrifugation was indicated in Fig.4. The purified particles were negatively stained, and examined with TEM [Fig.5(A), (B)]. No significant ultra-structure difference was observed between L1-VLPs (from Bac6L1 infection) and L1+L2-VLPs (from Bac6L1+Bac6L2 coinfection). Both types of VLPs were spherical particles composed of capsomeres. The particle diameter was approximately 50 nm, which is consistent with the diameter of isolated papillomavirus virions. Immunoelectron microscopy results showed that L1+L2-VLP reacted with both rabbit polyclonal antibodies to HPV-6 L1 and to HPV-6 L2 [Fig.5 insets(C), (D)].

 

Fig.4       The VLP band(indicated by arrow) in cesium chloride gradient ultracentrifugation

 

Fig.5       Electron microscopy and immunoelectron microscopy analysis of VLPs

A, B, VLPs purified from Sf9 cells infected with Bac6L1 and with Bac6L1 + Bac6L2, respectively. The insets C, D, immuno-electron microscopy analysis of the VLPs from B with antibodies to HPV6 L1 and L2, respectively.

 

To further characterize the VLPs composition, SDS-PAGE and Western blot analysis were carried out. As Fig.6 demonstrated, both L1-VLP and L1+L2-VLP contained L1 that was immunoreactive with H6C6 [Fig.6(A"), (B")], while L1+L2-VLP contained additional L2 that specifically reacted with HPV-6 L2 antibodies [Fig.6(B'), (B")]. These results together with immunoEM showed that the L2 was incorporated into the L1-formed VLPs when both proteins were simultaneously expressed. Calculated from the SDS-PAGE gel, the molar ratio of L1 to L2 in L1+L2-VLPs was about 4:1 under experimental conditions.

 

Fig.6       SDS-PAGE and Western blot analysis of the purified VLPs

M, protein marker with relative molecular weight indicated; A, B, VLPs purified from Sf9 cells infected with Bac6L1 and with Bac6L1 + Bac6L2, respectively. N' (N=A, B) is Western blot of N detected with antibody to HPV 6 L2, and N" is Western blot of N detected with a mixture of antibody to both HPV 6 L1 and L2. Solid arrows show the bands of L1 protein and hollow arrows for L2.

 

As characterized by ELISA (Fig.7), the L1+L2 VLP was found to be reactive with both H6E51 and H6K57 (recognizing surface linear epitopes an conformational epitopes of HPV-6 L1 VLP, respectively[13]), demonstrating the L1+L2 VLP possessed HPV-6 L1 VLP immuno-reactivities.

 

Fig.7       L1+L2 VLP immuno-reactivity in ELISA

 

2.5   Effect of L2 protein on L1 protein expression and VLP assembly

To determine interaction between L1 and L2 during expression we co-infected Sf9 cells with Bac6L1 and Bac6L2 at a series of MOI ratio of Bac6L1 to Bac6L2 (RL1/L2), which was 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 with total MOI (=10) constant. As Fig. 8 showed, expression level of L2 increased as Bac6L2 MOI increased (i.e. RL1/L2 decreased), while L1 level increased initially, highest at RL1/L2=75:25 (about 0.8 fold more than at RL1/L2=100:0), then dropped as Bac6L1 MOI decreased (i.e. RL1/L2 decreased).

Fig.8       Expression level of L1 and L2 protein in the Sf9 cells co-infected by Bac6L1 and Bac6L2 with different infection ratio

Sf9 cells were co-infected at MOI=10 by Bac6L1 and Bac6L2 with different ratio indicated. In the SDS-PAGE gel image the bands of L1 and L2 are pointed out. The amount of L1 protein for ratio of 100/0 is arbitrarily taken as 1, other L1 and L2 bands are calculated, and the relative values of protein amount are shown in the upper column graph that is vertically corresponding to the gel lanes.To investigate whether L2 affects VLP assembly, we determined the amounts of L1-VLP and L1+L2-VLP parallel purified from the same amount of infected cells (3×108 cells in 10 flasks of 175 cm2). The amounts of L1-VLP and L1+L2-VLP purified each time were represented by L1 protein and were calculated from SDS-PAGE of the purified particles with reference to internal BSA standard (Table 2). Co-infection with Bac6L1 (MOI=10) and Bac6L2 (MOI=5) increased VLP production by 3-4 folds in comparison with Bac6L1 single infection at MOI of either 10 or 15.

 

Table 2   Production of L1-VLP and L1+L2-VLP from 3×108 infected cells in two parallel experiments

 

L1-VLP

L1+L2-VLP

L1-VLP / L1+L2-VLP

MOI

Bac6L1=10

Bac6L1=15

Bac6L1:Bac6L2=10:5

in parallel experiments

Amount (μg)

Experiment 1

101

/

466

1:4.6

Experiment 2

/

103

441

1:4.3

 

3    Discussion

Caparros-Wanderley et al.[11] analyzed the entire L1 nucleotide sequence of 17 clinical isolates of HPV-6 from the London area and found the most frequently observed substitutions are clustered into three discrete regions: R1 (nt 5920 – 6075), R2 (nt 6590 – 6670) and R3 (nt 7070 – 7230). All of the five synonymous substitutions were observed in both JL2+1 and XL2+1 (Table 1). In the three synonymous substitutions in L1 ORF, ACA8106598→ACT was reported in R2 region by Caparros-Wanderley, while GAA12937081→GAG and AAG13117099→AAA, belonging to R3 region, were not reported in Caparros-Wanderley’s work and may represent region characteristics. The missense substitution TA2005988C→TGC(Tyr67→Cys) in L1 ORF of XL2+1 was also not reported which falls into R1 region. We excluded cloning and sequencing error by selecting 3 colonies for one insert and by repeating 2 times for a sequencing reaction. A larger sample size is needed to get clearer knowledge about the nucleotide preference in HPV-6 late region.

In contrast to the unique band specific to L1 in the Western blot detection of cell lysate protein [Fig.2 (A2')], L2 showed several faint bands in addition to the most intense band at 72.0 kD position [Fig.2 (B2')]. The bands were specific to L2 protein when compared with the corresponding positions in the control (non-recombinant baculovirus infected cell lysate). Together with the SDS-PAGE result [Fig.2 (B2)], in which a clear protein band existed at 72.0 kD position, we concluded that the multiple faint bands for L2 immunodetection was possibly due to L2 protein degradation, posttranslational modification, or aggregation with Sf9 cell endogenous protein. Similar results were reported for HPV-33 L2 expression in insect cells[14]. It’s worthy to note that the L2 molecules incorporated into L1+L2-VLP possessed a constant apparent molecular weight (Mr) of 72 kD [Fig.6(B)], which is higher than Mr deduced from its amino acid sequence (about 51 kD).

Papillomavirus L1 is able to self-assemble into VLPs when synthesized in eukaryotic cells, but not in E. coli. L1 synthesized in E. coli assembled into VLPs only after complex process of denaturing and renaturing, however, the assembling efficiency was very low (0.02% – 0.04%)[15]. The ability to self-assemble into VLPs is different among HPV L1 varients. It was known that HPV-16 L1 variants differed greatly in the self assembling ability[16], even lost the ability[17]. VLP formation efficiency is mainly determined by L1 amino acid sequence. However, the effect of necessary aa and their space situation on VLP formation is still remained to further investigation.

The molar ratio of L1 to L2 in the purified L1+L2-VLP was 4:1 with L2 composition in the VLP higher than that in virions isolated from biopsies(≈10:1)[18]. The difference was possibly related with the lack of interaction between capsid proteins with virus genome DNA in VLP, thereafter lack of assembly constraint. The MOI ratio of L1 to L2 recombinant baculovirus might also account for the difference. We used each MOI of 10 because L1 and L2 expression level was high and approximately equal to each other under this condition, and L2 composition in L1+L2-VLP varied only a little when MOI ratio differed. The major part taken by L1 in naturally occurring HPV particles or recombinant VLPs (for example, 100% in L1-VLP) reflects that L1 plays a ‘scaffold’ role and L2 plays a ‘staff-in’ role in virion assembly. In the VLP formation L1 is essential while L2 is optional. L2 is able to auto-incorporate into L1 VLP at a constant ratio. In this study we found that L2 protein enhanced L1 protein expression and L1 assembly into VLP.

In this article, we cloned two HPV-6 late region sequences from Chinese patients, produced HPV-6 VLPs from Sf9 cells by expression of L1 alone and coexpression of L1 and L2, and investigated the profiles of co-expression of L1 and L2. In particular, the L1+L2 VLP greatly mimics naturally occurring HPV virions (non-enveloped). The production of VLPs in large quantity is a first step towards an understanding of the viral assembly, the identification of the HPV receptor, the analysis of the mechanism of infection, and possibly the exploitation of DNA/gene delivery approach based on the VLPs. Such recombinant HPV capsids are also expected to be used as efficient vaccines against HPV[19]. In addition, they can be used to screen infected individuals for the presence of antibodies recognizing conformational epitopes on viral capsids.

 

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