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1 Department of Microbiology, Kobe Institute of Health, Kobe, Hyogo 650-0046, Japan
2 Department of Urology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan
3 Japanese Foundation for AIDS Prevention, Tokyo 105-0001, Japan
Correspondence
Yoshiaki Yogo
yogo-tky{at}umin.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AB242239–AB242255.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Sero-epidemiological surveys conducted in various countries have since demonstrated that this virus is ubiquitous in humans (Knowles, 2001). Infection most frequently occurs asymptomatically during childhood (Knowles, 2001). After the primary infection, BKPyV persists in renal tissue (Heritage et al., 1981; Chesters et al., 1983). The renal BKPyV becomes reactivated particularly in immunocompromised patients, and progeny viruses are excreted in urine. In renal transplant patients, BKPyV causes renal dysfunction, such as BKPyV-associated nephropathy (de Bruyn & Limaye, 2004).
BKPyV is the only primate polyomavirus that has subtypes distinguishable by immunological reactivity (Knowles, 2001). Jin et al. (1993) proposed genomic typing based on the nucleotide variations in a VP1 gene region that encodes aa 61–83. Using this variable region, the pattern of distribution of BKPyV subtypes has been studied in several countries including England, Italy, Tanzania, USA and Japan (Jin, 1993; Jin et al., 1993, 1995; Agostini et al., 1995; Di Taranto et al., 1997; Baksh et al., 2001; Takasaka et al., 2004). The results of these studies have indicated that (i) subtype I predominates in all geographical regions; (ii) subtype IV occurs at lower rates; and (iii) subtypes II and III rarely occur. In addition, subtype I has been subdivided into three subgroups named Ia, Ib and Ic based on nucleotide variations within the typing region and these subgroups were postulated to be associated with human populations (Takasaka et al., 2004).
The BKPyV genome has a transcriptional control region (TCR) between the origin of replication (Ori) and the start site of the late leader protein (agnoprotein) (Seif et al., 1979). The BKPyV TCR readily undergoes rearrangements of DNA sequences during passage of virus in cell culture (Yoshiike & Takemoto, 1986; Hara et al., 1986; Rubinstein et al., 1987). Therefore, the TCRs of BKPyV isolates obtained by viral culture could contain alterations introduced in vitro. In contrast, those obtained by molecular cloning or PCR should represent naturally occurring BKPyV TCRs. An analysis of BKPyV TCRs isolated using the latter method thus revealed that naturally occurring BKPyV TCRs have a common structure named the archetype (Moens & Rekvig, 2001).
The question arose as to why a similar pattern in the distribution of BKPyV subtypes has been established in different human populations. Among various possible factors affecting the proportion of the four subtypes of BKPyV in human populations is a potential difference among the subtypes in growth efficiency in human cells. Although this possibility could have been examined experimentally, to our knowledge, no comparative study of the growth capacity of various BKPyV subtypes has been conducted so far. In this study, we compared the growth capacity of two subtypes, the major (I) and the second major subtype (IV) in human kidney tubular epithelial cells recently shown to support the productive infection of a BKPyV strain with a rearranged TCR (Low et al., 2004). Although BKPyV strains with archetypal TCRs exhibited slow viral growth in vitro (Sugimoto et al., 1989), we hoped that long-term cultivation of the transfected cells in growth medium followed by titration of intracellular BKPyV using haemagglutination activity (HA) and PCR would allow the detection of viral growth.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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BKPyV DNA clones.
The origins of the recombinant BKPyV DNAs used in this study are shown in Table 1. BKPyV DNAs were cloned at the BamHI site within the VP1 gene. The recombinant plasmids were prepared by using a Plasmid Midi kit (Qiagen).
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. A recombinant plasmid in which a full-length BKPyV DNA was inserted at the BamHI site was digested with a combination of two restriction enzymes (SwaI and EcoT22I) cleaving the BKPyV at single sites but not cleaving the vector plasmid (pUC19). The two resultant fragments, one (E fragment) carrying most of the early region of the BKPyV genome and the other (L fragment) carrying the entire late region, part of the early region, the Ori, the TCR, and the vector pUC19 were isolated by the standard method (Sambrook et al., 1989). The E fragment derived from strain THK-9 and the L fragment from strain KOM-7 were ligated to generate a chimeric recombinant plasmid, pTHK/KOM. Similarly, (i) pKOM/THK containing the E fragment of KOM-7 and the L fragment of THK-9, (ii) pTW/THK containing the E fragment of TW-3 and the L fragment of THK-9, and (iii) pTHK/TW containing the E fragment of THK-9 and the L fragment of TW-3 were constructed. All recombinant BKPyV DNAs were fully sequenced to confirm not only that they had the exact designed construct, but also that no nucleotide changes were introduced during genetic manipulation.
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)]. After the addition of 800 µl serum-free OPTI-MEM I to cells grown on a 35 mm diameter dish and rinsed twice with 2 ml serum-free OPTI-MEM I, the Lipofectamine-BKPyV DNA mixture was loaded onto cells. Cells were cultured at 37 °C for 5 h, and then the medium was replaced with growth medium. After a change of medium at 24 h after transfection, cells were continuously cultured in growth medium with transfers at split ratios of 1 : 2 or 1 : 4 (only for the first passage). Aliquots of the cells were harvested on days 22, 35, 45 and 58 after transfection, and HA titration was performed as described below.
HA assay.
Transfected cells in a 25 cm2 flask were resuspended in 1 ml 1 mM Tris/HCl, pH 7.5, containing 0.2 % BSA, and frozen and thawed. The lysate was treated with 50 µg neuraminidase (Type V; Sigma) ml–1 at 37 °C overnight, incubated at 56 °C for 30 min and centrifuged at 1500 r.p.m. (RS-4 rotor) for 10 min at 4 °C. The resultant supernatant (designated ‘virus sample’) was serially diluted in a 96-well round micro-plate (Costar) in 50 µl PBS containing 0.2 % BSA. Following incubation at 37 °C for 1 h, 50 µl 0.5 % human O erythrocytes (provided by the Hyogo Red Cross Blood Center in Japan) were added and allowed to settle at 4 °C for 3 h. The HA titre was defined as the reciprocal of the greatest dilution of the virus suspension with which complete HA was observed.
Extraction of viral DNA.
Viral DNA was extracted from virus samples (see above) by the Hirt procedure with some modifications. Briefly, 0.5 ml of virus sample was mixed with 1.5 ml 10 mM Tris/HCl, pH 7.5, 0.8 % SDS, 14 mM EDTA, and then with 0.5 ml 5 M NaCl. The mixture was left standing at 4 °C overnight and centrifuged at 15 000 r.p.m. (TMA-2 rotor) for 30 min to remove high molecular-weight cellular DNAs. The resultant supernatant was digested with 20 µg proteinase K (Takara Shuzo) ml–1 at 37 °C for 1 h and treated once with phenol and once with a chloroform-isoamyl alcohol (24 : 1). DNA was recovered by ethanol precipitation and dissolved in 50 µl distilled water.
PCR.
The 287 bp typing region and the TCR were amplified from viral DNA by PCR using ProofStart DNA polymerase (Qiagen). The 287 bp region spanned nt 1650–1936 in the BKPyV (Dunlop) genome (GenBank accession no. NC_001538
[GenBank]
) and contained the entire effective sequence within the 327 bp typing region (Jin et al., 1993). Primers used to amplify the typing region were 327-1PST and 327-2HIN, and those used to amplify the TCR were RR-1PST and RR-2HIN (Takasaka et al., 2004). The total reaction volume of 50 µl contained 2.5 µl crude viral DNA, 1.25 U ProofStart DNA polymerase (Qiagen), 200 µM of each dNTP, 0.5 µM primers and a PCR buffer supplied by the manufacturer. After enzyme activation at 95 °C for 5 min, the amplification reaction was performed for 30 cycles. The cycle profile was 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min. Both activation and amplification were carried out in a Thermal Sequencer (Asahi Techno Glass Corporation). The amplified fragments were cloned as described previously (Takasaka et al., 2004), and recombinant plasmids obtained were prepared using a Plasmid Mini kit (Qiagen).
Sequencing.
Purified plasmids were used for a cycle sequencing reaction set up using the DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences). Primers used for sequencing the VP1 region and TCR were the T3 and T7 promoters (Toyobo). Primers used for sequencing the entire genome are shown in Table 2. The primers were added to a final concentration of 0.25 pmol µl–1 in a final reaction volume of 20 µl. The reaction profile was 25 cycles of 30 s at 96 °C, 15 s at 50 °C and 60 s at 60 °C. The reaction was terminated at 4 °C. Cycle sequencing products were purified on Centri-Sep columns (Princeton Separations). DNA sequencing was performed using an automated sequencer (ABI Prism 373S DNA sequencer; Applied Biosystems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). TW-3 and TW-3a were derived from the same urine sample, but differed by a single nucleotide within the agnogene, causing a change from Leu to Arg at aa 8. Transfected HPTE cells were allowed to grow in growth medium with occasional splits when the cells became confluent. About 8 weeks after transfection, cytopathology, noted as the detachment of cells from the plastic surface, was observed in the cells transfected with all subtype I isolates. However, no obvious signs of cytopathology were detected during the cultivation of cells transfected with subtype IV isolates (KOM-7, TW-3 and TW-3a).
Cells were harvested on days 22, 35, 45 and 58 after transfection and assayed for HA as described in Methods. The data can be summarized as follows (Table 3). Although a low level of HA was detected on day 22 in cells transfected with TW-8 belonging to subtype I, significant titres generally began to be detected in the cells transfected with subtype I isolates on day 35, and the titres peaked on day 58. In contrast, among the cells transfected with subtype IV isolates, HA was detected only on day 58 in the cells transfected with KOM-7 and not detected throughout cultivation in the cells transfected with TW-3 and TW-3a.
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Characterization of BKPyVs that replicated in HPTE cells
The 287 bp VP1 region contains several polymorphic sites that can be used for the discrimination of not only subtypes of BKPyV but also strains belonging to the same subtype (Jin et al., 1993; Takasaka et al., 2004) (Table 4). To confirm that the 287 bp sequences recovered from transfected cells were identical to those of the BKPyV strains used for transfection, we cloned the 287 bp VP1 regions PCR-amplified from HA-positive cell cultures and sequenced two representative clones for each cell culture. Although the two resultant sequences were usually identical, they sometimes differed by a single nucleotide mismatch, probably due to errors during PCR; in such cases, we sequenced a third clone to obtain a consensus sequence. The consensus sequences thus obtained were compared with the 287 bp sequences of the BKPyV DNA clones used for transfection, and it was found that, without exception, the 287 bp sequences from HA-positive cell cultures were identical to those of BKPyV DNAs used for transfection.
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). TCRs detected in cells transfected with Dik or KOM-5 (see below) could be distinguished by this single nucleotide polymorphism.
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In most cell cultures transfected with subtype I BKPyV DNAs with a single exception (see below), archetypal TCRs were most frequently observed on both days 35 and 58, with only the occasional detection of rearranged TCRs having a duplication or deletion or both (nucleotide sequences of these rearranged TCRs were deposited in the database) (Table 6). Furthermore, the archetype was also the major TCR in the HA-positive cell culture transfected with a subtype IV strain (i.e. KOM-7) (Table 6).
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), suggesting that a virus with archetypal TCRs initially grew in HPTE cells, and that during subsequent growth of this virus, variants with rearranged TCRs were generated.
Growth of chimeric BKPyV DNAs in HPTE cells
To clarify which portion of the BKPyV genome determines the capacity for growth in HPTE cells, we constructed a chimeric BKPyV DNA that was composed of the L fragment of a subtype I strain (THK-9) and the E fragment of a subtype IV strain (KOM-7 or TW-3) (see Methods and Fig. 1). Fragment E contained most of the early region including the central region of the large T antigen (LTag) gene, while L fragment contained the entire late region (i.e. the agnogene and VP1–3 genes), the 5'- and 3'-terminal regions of the LTag gene, the Ori, the TCR and the vector pUC19. We also constructed a complementary chimeric BKPyV DNA that was composed of the L fragment of KOM-7 or TW-3 and the E fragment of THK-9. According to the protocol described above, the chimeric BKPyV DNAs and the parental viral DNAs (THK-9, KOM-7 and TW-3) were individually used to transfect HPTE cells, and the transfected cells were allowed to grow in growth medium with occasional splits. Cells were harvested on days 22, 35, 45 and 58 after transfection and assayed for HA as described above. The data can be summarized as follows (Table 7).
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We partially sequenced viral DNAs recovered from the HA-positive cells transfected with chimeric BKPyV DNAs. Based on the sequence data (not shown), we confirmed that (i) they carried the L fragment derived from a subtype I strain (THK-9) and the E fragment derived from a subtype IV strain (KOM-7 or TW-3), and (ii) BKPyV strains with the archetypal TCRs replicated in HPTE cells.
From the findings described above, we concluded that the L fragment is mainly responsible for the low growth capacity of subtype IV strains in HPTE cells, with some involvement of the E fragment.
Genome regions critically involved in the inefficient growth of subtype IV strains
The L fragment encompasses a wide area of the BKPyV genome, including the entire late region, part of the early region, the Ori and the TCR. Nevertheless, the area critically involved in the inefficient growth of subtype IV BKPyV in HPTE cells could be restricted based on complete nucleotide sequences of subtype I (THK-9) and IV (KOM-7 and TW-3) BKPyV strains used to construct the chimeric BKPyVs (see Table 1 for the GenBank accession nos of the complete DNA sequences of these strains).
(i) The structures of the Ori and the TCR are completely identical, or essentially identical, between subtype I and IV BKPyV strains used in the present study.
(ii) The nucleotide sequences of the 5'- and 3'-terminal regions of the LTag gene included in the L fragment were translated into amino acid sequences, which were compared between subtype I and IV BKPyV strains. The N-terminal portion of the LTag was identical between subtype I and IV BKPyV strains, but the C-terminal region differed by a few amino acids.
(iii) The nucleotide sequences of four late genes (i.e. agnoprotein and three capsid protein genes) were translated into amino acid sequences, which were compared between subtype I and IV BKPyV strains. In the agnoprotein, there was no common difference between subtype I and IV BKPyV strains, although unique amino acid changes occurred in individual strains. In the major capsid protein VP1, 17 aa differed between subtype I and IV BKPyV strains. In the minor capsid proteins, VP2/VP3, 12 aa differed between subtypes I and IV (one was unique to VP2 and 11 were shared by VP2 and VP3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The distribution of various BKPyV subtypes has been studied in several geographical areas (see Introduction). Generally, subtype I occurs most frequently, followed by subtype IV, with subtypes II and III rarely detected. As HRE cells probably represent cells in which BKPyV replicates in vivo (Randhawa et al., 1999), the conclusion of the present study that subtype I BKPyV replicates in HRE cells more efficiently than subtype IV BKPyV suggests that the growth capacity of BKPyV determines the proportion of subtype I to subtype IV in human populations. Nevertheless, to clarify whether growth ability in HRE cells is generally related to the distribution of BKPyV subtypes in human populations, two other subtypes (II and III) of BKPyV remain to be examined for growth efficiency in HPTE and HRE cells.
We found that chimeric BKPyVs constructed from the E fragment derived from subtype IV strains and the L fragment from subtype I strains exhibited enhanced growth capacity in HPTE cells as compared with the parental subtype IV BKPyV strains. The growth capacity of each of these chimera, however, was less than that of the parental subtype I strain (THK-9) used to construct them. Furthermore, complementary chimeric BKPyVs constructed from the E fragment derived from subtype I and the L fragment from subtype IV showed little capacity for growth similar to subtype IV strains. Altogether, we concluded that the L fragment is mainly responsible for the low growth capacity of subtype IV strains in HPTE cells, with some involvement of the E fragment. Furthermore, based on comparisons of the subtype I and IV strains used to construct the chimeric BKPyVs, we concluded that amino acid changes occurring in the C-terminal portion of the LTag and throughout the three capsid proteins (VP1–3) are candidates for amino acid changes involved in the low growth capacity of subtype IV in HPTE cells. It may be of interest that included among these amino acid changes are those within the capsid protein VP1 that potentially affect virus entry into cells, thereby altering the efficiency of viral growth (Gee et al., 2004).
It has been reported that the BKPyV TCR readily undergoes rearrangements of DNA sequence during passage of virus in cell culture (Yoshiike & Takemoto, 1986; Hara et al., 1986; Rubinstein et al., 1987). All BKPyV strains used in the present study had archetypal TCRs. Interestingly, these archetypal TCRs were essentially conserved in the course of viral growth in HPTE cells. The stability of archetypal TCRs may depend on the strains of virus and the cells in which BKPyV was propagated. In this study, we found that TW-8 belonging to subtype I/subgroup Ic more readily produced variants with rearranged TCRs. According to published sequence data (Takasaka et al., 2004), the TCR (Seq-9) of TW-8 had a single nucleotide change as compared with that (Seq-7) of most strains belonging to subgroup Ic. At present, it is unclear whether this mutation affects the stability of the TCR. It appears that archetypal TCRs are rather stable during viral growth in cultured target cells, as HPTE cells probably represent target cells where a productive infection of BKPyV occurs in vivo (Randhawa et al., 1999). Actually, we recently observed that the TCRs of BKPyV changed rapidly during the viral growth after transfection of renal epithelial cells obtained from whole human kidney (unpublished data).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 November 2005;
accepted 9 March 2006.
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