Genomic Function and Variation of Human Polyomavirus BK (BKV)

Reviews in MEDICAL VIROLOGY VOL. 6: 201-214 (1996)

Genomic Function and Variation of Human Polyomavirus BK (BKV) Li Jinx and P. E. Gibson Virus Reference Division, Central Public Health Laborafory, 6 1 Colindale Avenue, London NW9 5HT

GENERAL HISTORY OF BK VIRUS

BK virus was first isolated from the urine of an immunocompromised renal transplant patient in England in 1971.’ Meanwhile, the isolation of another human papovavirus was reported: JC virus was recovered from the brain of an American patient with Hodgkin’s disease and progressive multifocal leucoenkephalopathy (PML).’ Morphologically the two viruses belong to the polyomavirus genus, were classified as human polyomaviruses and were recognized by the International Committee on Taxonomy of Viruses as new species of the genus polyomavirus in 1976.3 They are antigenically different from one another and from all other animal polyomaviruses although, using hyperimmune antisera, cross- reactions occur. BK virus (BKV), JC virus (JCV) and simian virus 40 (sV40) share a minor, common antigenic determinant which is exposed on the surface of virions.4p7

Both BKV and JCV circulate in a large proportion of the population worldwide. Like polyomaviruses of ani- mals such as murine polyomavirus (Py), they persist after acute infection but rarely produce recognisable disease. Immunosuppression due to disease or chemotherapy can lead to reactivation of the persistent virus followed by prolonged excretion, the site of shedding being the kidney epithelium.’ There is substantial evidence for an aetiological association between both BKV and JCV and ureteral stenosis in renal transplant (RT) recipients, and haemorrhagic cystitis in bone marrow transplant (BMT) recipients.‘,’’ There is also the possibility that infection with these viruses might contribute to other complica- tions that are known to arise in immunocompromised groups e.g. respiratory tract disease and tumours.’~’

Genomic subtypes of BKV have been described and are found to circulate independently in the human

“Author to whom correspondence should be addressed.

Abbreviations used: BKV, BK virus; BMT, bone marrow transplant; HAI, haemaggalutination inhibition; HEK, human embryo kidney; IR, inverted repeats; JCV, JC virus; LL, late leader; NCR, non-coding control region; NF, nuclear factor; NLS, localisation signal sequence; NTS, nuclear transport signal; PML, progressive multifocal leuco- encephalopy; Py, pol yomavirus of mice; RT, renal transplant; SV40, simian virus 40; TI’, true palindrome.

population.ll,lz Although the reactivation of different subtypes may require different levels of immunosuppression, it has not been possible to associate BKV subtypes with particular clinical symptoms or to particular groups of immunocompromised

Molecular biological investigations have provided valuable conceptual information on the genomic structure and the function of viruses in general, in the mechanism of viral transformation of cells, and on the mode of viral replication during lytic infection. The weIlLdefined, simple structure of polyomaviruses has also made them attrac- tive models for studying the complex biology of eukaryotic cells and the mechanisms of eukaryotic DNA rep1i~ation.l~ Furthermore, s v 4 0 has been used as a vector in recombinant DNA studies. BKV and JCV have also been recognised as powerful tools in molecular studies.

This article reviews the genomic organisation and molecular biology of BKV, and relates this to its infectivity and pathogenicity (including tumour formation). This review includes the genetic basis for BKV heterogeneity, since the molecular epidemiology of BKV infection may soon become an important field of study.

ORGANISATION AND FUNCTION OF THE VIRAL GENOME

All polyomaviruses have a closed circular dsDNA genome with a M, of about 34-35 x i ~ ~ . ~ ~ - ~ ~ The complete DNA sequences of BKV contain 5153 bp for strain DUN,’5 4963 bp for strain MM17’18 and 5098 bp for strain AS.” The predicted amino acid sequences of the BKV share 73% homology with those of S V d 9 and 75% with those of JCV.’’

From the nucleotide sequences, the BKV genome can be functionally divided into three regions (Figure I). The coding regions for the two ’early’ proteins (T and t antigens) and the four ’late’ proteins (agno and capsid virion proteins, VP1-3) are separated by the non-coding region (NCR) which contains the transcriptional control elements for both ’early’ and ‘late’ gene expression, as well as containing the viral origin of repl i~at ionl~,’~

202 LI JIN AND P. E. GIBSON

T-Ag

Figure 1. Diagramatic representation of the gene organisation in BKV genome. The map coordinates are orientated clockwise from the EcoR I cleavage site at 0.0 map units. The inner arcs indicate the early and late regions and the direction in which the mRNAs are transcribed. The origin of replication is indicared at 0.67 map units and the noncoding control region is located outside the genome of BKV. Coding regions are depicted by thick-black arrows for early proteins (t-Ag and T-Ag) and for late proteins (Agno, VPI. VP2 and VP3).

The non-coding control region The sequence and organisation of the archetypal BKV control region are shown schematically in Figure 2. The origin of replication (ori) is located within the start codons of the T/t antigens and the putative agnogene and the control region lies between regions of different polarity. Its sequences (nt 1-139) contain a number of inverted repeats (IR) or dyad symmetries, a true palindrome (TP) and a 20-bp AT block. One of the IR (IR23), contains a second T arkigen binding site which appears to function as the site from which bidirectional replication of the viral DNA is initiated.22 The pentanucleotide sequence, 5’-(G>T)(A>G)GGC-3’, has been proposed as the recognition sequence for the T-Ag of SV40. Similar T-Ag binding sites in BKV are located in the TP region at nucleotides (nt) 5 5 to 51 and nt 67 to 63 (opposite polarity, GCCTC), which together act as the T-antigen binding site I. The sequence 5’-AAGGC-3’ (nt 138-142) has also been identified as the consensus sequence for another T-antigen binding site (site 111) by DNase foot-

Deletion mutants of the BKV non-coding control region have been used to define the BKV origin of replication in several cell types. Such studies have indicated that the BKV minimal origin of replication includes an inverted repeat, the T-Ag binding site 11, and a 20-bp A T block at nt 120-139. However, the particular sequences that are required to enable the replication of a given BKV strain vary depending both on the cell type and the T-Ag, probably reflecting differences in cellular factors and the viral T-Ag function. The BKV origin of

replication shows similarities to, and differences from, that of SV40 and Py.25

The second part of the non-coding control region, which forms the transcriptional regulatory region (potential cis-acting elements), has been divided arbitrarily into three transcription factor binding regions called blocks P (68 bp), Q (39 bp) and R (63 bp) and the late leader (LL, 63 bp) region. The altered sequences are related to the existence and function of discrete transcription/ translation e l e r n e n t ~ . ~ ~ , ~ ~

The binding of cellular factors to the BKV non-coding control region has been analysed by DNase I footprint- ing. Potential cis-acting elements have been described for the archetypal strain (BKV-WW) and include binding sites for the nuclear factors specific for BKV (NF-BKV), and for the host cell transcription factors Sp l and L 1 (Figure 2) .27

NF-BKV is a member of the nuclear factor I (NFl) family and is specific for BKV. The consensus NF-BKV binding site, TGGAATIAGICCITAITGCCAAA, is found in block P, in block R (where there two sites) at the junction between blocks P and Q, and between the right-hand junction of block R and the coding sequence for the agnoprotein (Figure 2). These sites are recognised by the same or a closely related protein. Similar sites are present in other strains but in different numbers (Figure 3). These NF-BKV sites are functional elements of the BKV enhancer and are important for growth in culture.’’

The transcriptional activator Spl binds to the consensus sequence GGGGCGGGGT, which is located at the

GENOMIC FUNCTION OF BKV 203

A.

CAT TTTTGCAAAA ATTGCAAAAG AATAGGGATT TCCCCAAATA GTTTTGCTAG GCCTCAGAAA 60

'R l9 IRlO I 11 TP17 c- - -+-a

AAGCCTCCAC ACCCTTACTA CTTGAGAGAA AGGGTGGAGG CAGAGGCGGC CTCGGCCTCZ 120 I- ._

NF In27 I11 'R23 - r p 6 8

TATATATTAT N Q A M A A A G GCCACAGGGA GGAGCTGCTA ACCCATGGAA TGTAGCCAAA 180 AT

r Q 3 9 NF CCATGACCTC AGGAAGGAAA GTGCATGACT GGGCAGCCAG CCAGTGGCAG TTAATAGTGA 240

- Spl P R 8 3 NF AACCCCGCCC CTAAAATTCT CAAATAAACA CAAGAGGAAG TGGAAACTGG CCAAAGGAGT 300

NF p LL63 L1 GGAAAGCAGC CAGACAGACA TGTTTTGCGA GCCTAGGAAT CTTGGCCTTG TCCCCAGTTA 360

NF r 'Agno NF AACTGGACAA AGGCCATGGT TCTGCGCCAG CTGTCACGAC AAGCTT

B.

19 10 17 27 23 20 68 39 63 ~ / t ~cA-HTH TP H IRI IR I ATH

5 17 1 3 P -1 R E + A g n o

Figure 2. Nucleotide sequences of the non-coding control region of archety e BKV-WW (A) and the relative archetype schematic diagram (B). The number system is that used by Seif et a h 4 The start codons for the Th-ag and the agnogene are indicated by thin arrows. The locations of the sequences IR, TP and AT are underlined, and blocks P, Q, R and late leader (LL) are indicated by thick arrows. Putative binding sites for transcription factors are overlined and three T-Ag binding sites are also indicated with Roman numerals. The direction of arrows shows the polarity. The GenBank accession number of the sequence is M15987. CAT: opposite polarity of the start code for the early proteins. Numbers: sequence lengths in bp.

junction of blocks Q and R (Figure 2); Spl may act in conjunction with other transcription factors (NFl and API) which also bind to the BKV enhancer The Spl site is not found in strain BKV-DUN and is partially deleted from strain BKV-PT.

A late proximal region in strain BKV-WW which overlaps the start codon for the agnoprotein is referred to as LI . The sequence of region .LI which serves as a protein-binding site is TGGCCTTGTCCCCAG. Strain BKV-AS does not contain the LI site (Figure 3) but this deletion does not affect early gene expression.18p32 Studies on SV40 have shown that L 1 overlaps the initial part of the 'late' coding region so that its binding to this region affects 'late' gene e x p r e ~ s i o n . ~ ~ It is not clear if the function of L1 has any bearing on the serological variation that has been documented among BKV isolates in haemagglutination inhibition (HAI) tests, since the 'late' structural proteins are responsible for the HA activity of p o l y ~ m a v i r u s e s . ~ ~ ~ ~ ~

There are two predicted binding sites for the transcription factor APl in strains BKV-DUN and BKV-PT. In these strains, the sites cross the junction between adjacent

P blocks. Such contiguous blocks are not found in other viral strains (Figure 3). APl binds to the sequence TGACTCA.36 Such binding sites appear to have been created at the time the P block was tripled in BKV strains DUN and PT (Figure 3). The artificial addition of one or two synthetic AP1 binding oligonucleotides to the unique Bsu36I site in the block P of the archetypal strain BKV-WW resulted in a large increase in early gene

On the other hand, the addition of a further one, two or three NFl binding sites did not increase 'early' gene activity. This demonstrates that the inserted APT sites can complement the sequences of the non- coding region normally present in BKV-WW, to form a functional unit capable of driving high levels of BKV early gene expression. Thus a number of independently isolated BKV-WW like strains may have reduced growth potential because of deletions in their promoter-enhancer regions.

analysed the early promoter and enhancer of strain BKV-PT by deletion analysis. Their data suggested that the BKV 'early' promoter and enhancer are overlapping elements. The minimal BKV

26,27,3 7

Deyerly et

204 LI ]IN AND 1’. E. GIBSON

Figure 3. Comparison of the noncoding control region of BKV strains. The letters CAT within the open box to the left represent the initiation codon (opposite polarity) for the early proteins. To the far right is the ATG initiation codon for the agnoprotein. €Iomologous sequences are aligned vertically and shaded similarly. Regulatory blocks are indicated by rectangles and are identified (ori, A/T, P, Q, R, LL) in the archetype. Numbers shown are sequence lengths in base pair. The GenBank accession numbers of these sequences are shown in the brackets after the references.

early promoter is composed of the sequences TP-IR- IR-AT plus the early portion (18 bp) of the first block P68 (Figure 2, Figure 3). Markowitz and DynanL6 proposed division of the control region of the WW strain into three blocks labelled P, Q and R. These blocks contain 68, 39 and 63 bp respectively. Strain BKV-AS contains the same arrangement, and these sequences probably form the early promoter for these two viruses. The enhancer for strain BKV-PT includes a triplicated P block with a 18 bp deletion in the middle P block (P68-P5O-P68) and block Q39. BKV-WW, BKV-AS and other strains have a single enhancer element located in block Q39 and a 63 bp block known as R63 (Figure 3) whose function is not very clear. By chloramphenicol acetyltransferase assay, the level of BKV-WW enhancement as represented by the construct P68-Q39-R63 [see Figure 2 for the sequence) is about half that of the corresponding BKV-PT constructs (P68-P50- P68-Q39). A chloramphenicol acetyltransferase construct containing a single P68 unit, but lacking block R63, showed only 3% of the activity of BKV-PT. Therefore, block R63 in BKV strains WW and AS (Figure 3) might substitute for block P68 to yield full enhancer activity, but this has not been tested experimentally.” These sequence variations in the noncoding control region of BKV strains may contribute to the different biological activities of the virus.

The ’late’ promoter is able to direct late-direction RNA synthesis (opposite to the direction for the ’early’ RNA synthesis) and functions to enhance the expression of ’late’ proteins. Cassill and Subramani3’ found that the late promoter of strain BKV-PT is contained entirely within the enhancer for the ’early’ promoter of the virus, that is the P68-P5O-P68 repeat and the C element. Thus, constructs with deletions in the ’early’ enhancer domain, when tested in chloramphenicol acetyltransferase assays, resulted in a two- to three-fold decrease in the activity of ’late’ gene expression. The transcriptional start sites of

the BKV-PT ’late’ promoter (‘late‘ mRKA start sites) are located in Block Q39. In strain BKV-PT the overall structure of the ’late’ promoter appears to be three SpI or Spl-like sites and three NF-BKV sites dispersed across 243-by. All these elements contribute to the ’late’ promoter activity, but the most important elements appear to be the NFI site in the third P68 repeat, and the Spl binding site in the ’late’ side of the control region. The Spl binding site is a 66-bp region that includes the ’late’ mRNA start sites. Different interactions between the transcription elements contained within the same region of DNA are responsible for modulating ’early’ and ’late’ transcrip t i ~ n . ~ ’ , ~ ’

The early region According to the full sequences published for the strains BKV-DUN and BKV-MM,15”7 the early region contains two ORFs both of which encode nonstructural proteins, namely the T and t antigens (T-Ag and t-Ag), and are transcribed prior to viral DNA synthesis. It is assumed that the same triplet ATG that is used for initiating t protein synthesis is also the start codon for the T protein. Hence both have identical amino termini.41p-‘4

The BKV T-Ag of M, 94 000 (695-aa for BKV-DUN) is encoded by two non-contiguous regions of the genome which become joined together by splicing of the mRNA.’l Some s t ~ d i e s ’ ~ , ~ ~ suggest that the T-Ag has an important role in gene expression. BKV can transform human embryo kidney (HEK) and brain cells in uilvo and produce tumours in uivo when injected into young hamster^.'^-^' Most of the tumours and transformed cells retain the intact ’early’ region of BKV DNA and express the antigens T and t, but strain BKV-MM is unable to express the ’early’ t-Ag, although it is still capable of inducing ependym~mas.~’

BKV can efficiently transform HEK and brain cells in culture (48,501. Anchorage independent growth of HEK


cells has been achieved bs7 a combination of infection with BKV D R A plus the human m oncogene.5' All the transformed cells retained the intact early region of BKV DNA and expressed the T and t antigens of BKV. Using recombinants, only those with this 'early' region were able to transform. Nakshatri et also presented evidence that BKV transformed cells (BHKZI and NiH3T3) expressing antisense T-Ag RNA lose their ability to grow and to maintain the transformed pheno- type. The domain of transformation was localised to the region of aa 356-384. These observations indicate that T-Ag is a transforming protein.

Although the non-coding control region of a virus can contribute to the tissue and host specificity, many of the observed differences in the lytic and transforming functions between BKV and sV40 appear to be due to variation in the biological properties of their T-antigens. This has been confirmed for certain functions of SV40," but similar studies on other polyomaviruses have not yet been carried out.

The nuclear localisation signal sequence (NLS) Lys- Lys-Lys-Arg-Lys is present in the SV40 T-Ag.'4,5' The T-Ag in sV40 is found predominantly in the nucleus where it is required for the initiation of viral DNA replication, and for the stimulation of cellular DNA synthesis. T-Ag synthesised by Py also regulates the synthesis of host mRNA and rRNA in addition to regulating viral transcription56. BKV T-Ag has also been shown to be capable of stimulating late transcription."

The BKV t-Ag of M,. 17 000 is translated as a 171-aa protein (in strain BKV-DUN) directly from a single ORF (Figure 1). Mutants vvithout the t-Ag region have reduced efficiency of transformation."," In this context, it should be noted that tumours induced by the t-Ag mutants of SV4O show enhanced metastatic potential and altered tissue specificity.'7,58

The late region The 'late' region of the BKV genome, like that of S V ~ C I and other polyoma viruses, codes for the structural proteins VPI, VP2, VP3 and the agnoprotein (Figure 1). BKV VP1 like that of SV40 is composed of 362 aa. All members of the polyomaviruses appear to share a common antigenic determinant on their major capsid polypeptide.19,59 The structural and biological functions of the VPI within the virus particle have been studied mainly in Py, and is composed of six isoelectric species, as is the VPI of SV4O."" The patterns of these six species in SDS-polyacrylamide gel electrophoresis showed that their pI values varied considerably between Py and sV40. This diverse pattern may be a common feature of the polyomaviruses.ol

Bolen ef nl."' reported that the VPI may serve at least three distinct functional roles, the first of which is structural. isoelectric species A was found to be an internal protein tightly associated with the virion chro- matin core, whereas the other VPI species (B-F) were found to be exposed on the surface of the virion. The VPI isoelectric species B, C and D are present in capsomere preparations, and species E and F may be

involved in the penton capsomere subunits of the virion. Thus, potentially five of the six VPI species perform a structural function for the virus.

The second role is exemplified by the V P I species A. Studies on sV40, suggest that the VP1 species A in the complexes maintains the chromatic core of the \+us particle in a compact form and increases the potential accessibility of the viral DNA to enzymes, including RNA polymerase, and so may affect the genetic expression of viral DNA.O@,CI

The third function deals with the ability of the virus to adsorb to mammalian cells such as guinea-pig erythro- cytes and mouse kidney cells. Three of the VP isoelectric species D, E and F are involved in such activities. The species D and F are present both in virions and empty capsid shells, whereas species E is present only in virions. These three species apparently function as viral attach- ment proteins that enable virus adsorption and haemagglutination. Immuno-precipitation studies suggest that species E is required for virus adsorption to specific cellular receptors and for the ability of the virus to infect mouse kidney and mouse embryo cells.6o SV40 has a NLS in the first eight aa (Ala-Pro-Thr-Lys-

Arg-Lys-Gly-Ser) of the VP1, excluding the initial methionine. In BKV isolates, the first seven aa of the VPI are identical to SV40, but glutamic acid replaces serine at position eight.1531s,62 A recent study using truncated constructs of polyomavirus VPI that lack the NLS sequence revealed that while the full-length VPI is expressed and localised in the cytoplasm, i t is not transported to the nucleus. By site-directed niuta- genesis it was shown that certain aa within the nuclear transport domain of the V P I protein are crucial for the transportation activity."

The first seven aa located in the VPI amino terminus, which are identical to the putative NLS, represent the DNA binding domain. This is required for the high affinity binding of VPI to DNA during virus assembly.63,c4 The assembly of polyomavirus occurs in the nucleus of the infected cell. While the Py VPI can be assembled in vitro and in via0 into capsid-like structures, in i l i h studies of the properties of the binding of VPI alone to viral DNA suggest that the minor capsid proteins VP2 and VP3 are involved in modulating the encapsidation of the viral minichromosome by VPI during virus assembly.CJ-c'l

The functions and positions of the two minor BKV proteins VP2 and VP3 within the structure of the virion are not well known. The proposed sequence for BKV VP2 contains 351 aa of which the carboxy terminal 232 aa are shared with VP3. Streuli and Griffin" first reported that the amino terminal methionine-glycine (Met-Gly) sequence which is present in the VP2 of all the polyomaviruses, including BKV and JCV, is the consensus sequence for myristyl modification. The myristyl- modified protein, the fatty acid component of which becomes membrane associated in the nucleus, is important for the transportation of the non-enveloped virus. Myristoylated VP2 is critical for several stages in the viral life cycle including entry into the cells, transport to

,~ I -


the nucleus, and uncoating. Hence, the myristoylation of VP2 is not an absolute requirement for infectivity, but facilitates efficient viral infection.67

The VP2 and VP3 of SV40 also carry a NLS near their carboxyl end (aa 317-323 in VPZ), with the sequence Pro-Asn-Lys-Lys-Lys-Arg-Lys.68 In the strains BK-DUN and BK-AS the analogous regions in VP2 are identical (aa 316-322) and have the sequence Pro-Asn-Gln-Lys- Lys-Arg-Arg.18,21 Because the ends of the VP2/VP3 ORF overlap, by 113 nucleotides, at the beginning of the VPI in SV40, this sequence must also be responsible for the migration of its VPI into the cell nucleus and for its accumulation. This NLS sequence is encoded by the same portion of the viral genome as VP2 and VP3, but is read in an alternative frame in VPI. It has been suggested that this NLS sequence allows the VP2 and the VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cytoplasm to the nucleus by interaction with VPI, which acts as a ~ a r r i e r . ~ ' . ~ ~

The NLS may be divided into two component parts: a nuclear transport signal (NTS), which is an entry signal that is recognised by nuclear pore receptor(s), and a nuclear retention signal which allows protein to accumu- late within the n ~ c l e u s . ' ~ A consensus sequence for the NTS, namely Lys-LyslArg-X-LyslArg (X = Lys, Arg, Pro, Val, Ala) has been proposed. Recent data obtained using microinjection have indicated that small nuclear proteins such as the VP2 and VP3 (Mr 38,000, 27,000) contain their own NTS and localize to the nucleus by a facilitated transport process, which is similar to the mechanism that has been described for large nuclear proteins.56

Based on the evidence of genetic and biochemical studies the agnoprotein of SV40 may play a late role in the lytic cycle, perhaps in the assembly of virions. Mutants which make no agnoprotein yield less virus and form smaller plaques than the wild type SV40. While they display abnormal perinuclear-nuclear localisation of VPI, the localisation of VP2 and VP3 is n ~ r m a l . ' ~ , ~ ~ This suggests that at least one function of the agnoprotein in SV40 is to enhance the efficiency of the perinuclear- nuclear localisation of VPI. This role of the agnoprotein may be important for maintaining the efficient packaging of the viral proteins so enhancing virus yields from infected cells and facilitating viral spread.'; Little has been reported about the agnoprotein of BKV. A NF binding site has been identified at the initial part of the agnoprotein gene of strain BKV-WW,74 suggesting that the agnoprotein may be involved in viral transcription.

HETEROGENEITY OF THE BKV GENOME

Defective genomes Defective BKV genomes are well-known by-products of viral multiplication during undiluted passage in cultured cells.75 In JCV, both the multiplicity of infection and the cell type have been shown to influence the degree of heterogeneity observed in the viral DNA sequences. The

extent of such heterogeneity is considered to be an intrinsic property of the viral

Defective BKV genomes have been found to have the following properties: (I) they contain re-arranged DNA and are non-infectious; (2) they cannot induce T-Ag synthesis or transform hamster cells, but interfere with the replication of infectious (3) they can replicate in the presence of the homologous infectious virus. I t has been proposed that complementation may occur when two defective DNA genomes coinfect the same cell, resulting in productive infecti~n.'~.'" Only full-length BKV genomes have been reported to be infectious, so that in order to produce virus stocks in tissue culture in which complete genomes predominate, viruses have to be passaged at limiting dilution."

I t has been questioned whether some of the shorter molecules which are present in preparations of heterogeneous viral DNA represent defective genomes or viable mutants. Early comparisons failed to detect any significant antigenic differences between viruses containing deletions and those without. This may have been due to the insensitivity of the assay system (fluorescent cell assay), to the use of polyclonal rather than monoclonal antibodies for studying virus neutralisation, and the ability of defective viral genomes to complement one another for virus growth. Experiments with JCV using modified genomes of cloned DNA transfected into eukaryotic cells produced defective Mechan- isms that generate defective genomes might be involved in the generation of new viable variants and, in this way, play an important role in the evolution of these viruses.

Non-defective genomes BKV DNA has been studied extensively by the con- struction of restriction endonuclease cleavage maps, by determining base sequence homology with other polyomaviruses, by hybridisation re-association kinetics, by filter blot hybridisation under conditions of controlled stringency and by electron microscopy.

Alterations in the non-coding control vegion Within its length of about 300-500 bp, the structure of the NCR varies widely among BKV strains. At least two types of control regions can be found among naturally occurring strains. O n e is found in strain BKV-WW, which is considered to have the archetypal structure for BKV since its sequences in the control region were obtained directly from the virus in urine, either by direct cloning or by PCR ampl i f i~a t ion . '~ -~~ The second is found in other strains, such as the BKV-prototype PT, DUN, MM and GS, which were subjected to repeated passages in cell culture before detailed characterisation was initiated. Partial duplications and/or deletions have been recognised in the sequences of the control regions of these latter strains.'*

Ever since the first isolation of BKV from urine, the genomes of different isolates have been found to be heterogeneous in size and in the distribution of recognition sites for restriction endonucleases. Some of the


A

The known Sources of length (bp) PCR template

M (bp) DUN PT

IR GS ww 56 57 J8 TI0 w2 C183 RI R2 R3 H8 D4

pFT-s/l

D8- I D8-2 DI 1 - 1 DI 1-2 D18-1 D 18-2 D 18-3 D 18-4 M (bp)

:V-

698

762 63 I 686

PC PC PC PC PC PC vc vc vc

Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine

- Figure 4a.

Figure 4. PCR product DNA: the results of amplifying the control region of BKV strains present in urine, in supernatants of viral cultures (VC) and in plasmid cultures (PC). The sizes of the fragments for some isolates for which there is already published information are presented for reference. Samples originally from the same patients were sub-numbered (e.g. D8-I, DS-2) and tested for comparison. All the samples in Part A were known to be subtype I of BKV, based on the subtype-specific sequences in VPI, whereas Subtypes 11, I11 and IV are in Part B.

differences are localised to the non-coding or regulatory Similarly, many such differences have

been found in the genomes of JCV strains. Most of these were in the noncoding sequences, and in the enhancer elements within these noncoding regions; they may therefore be active in influencing infectivity, host range and growth rate.76,88T89

Enhancer sequences obtained as amplified PCR products demonstrate the highest diversity, not only between polyomaviruses, but also between different strains of

schematic diagram of the regulatory region of some BKV

region. 73.83,86.87

BKV of blocks P68-Q39-R63-LL63 (Figure 2).”9,90r91 A

strains is shown in Fig 3. The BKV strains BKV-PT and BKV-DUN, which were independently isolated in cell culture, have identical block P repeat structures and BKV-DUN has a deletion of block Q39 in the non-coding regi~n.’’ ,~~ Strain BKV-WW, which was isolated by molecular cloning, does not contain sequence repeats. Strain BKV-17, also isolated by molecular cloning was shown to be identical in its regulatory region to strain BKV-DUN.26 Two antigenically distinct strains of BKV, namely AS and DB, both of which have been passaged extensively in tissue culture, do not possess a large randomly repeated structure within their regulatory

208 LI JIK AND P E. GIBSON

B

I

M (bp) SB IK SB69 SB73 SB72- I SB72- 1 I SB72- 12 SB7 1-6 SB7 1-7 SB7 1-9 SB71-1 1 SB7 I - I5 J1 53 D2 B7- 1 87-2 34 IV pIV-s/PT- I IV55 IV53 DB-8 DB-9 DB-I 1 J5 52 M (bp)

Subtype

I 1 I1 I1 I1 I1 I1 I1 I1 I1 I 1 I1 I1 I 1 I 1 I1 I 1 I1 111 IV IV IV 1v IV IV IV IV IV

Sources of PCR template

PC PC PC PC PC PC PC PC PC PC PC PC vc vc

Urine Urine Urine vc PC PC PC PC PC PC PC vc vc

Figure 4b

regions. Furthermore, strain DB is different from strain AS in possessing a 53-bp duplication in blocks P and

0iigonuc;eotide primers compiementary to the T antigen and to the VP2 regions of BKV, which are conserved amongst the BKV strains, have been used to amplify the h C R of BKV, by PCR.92 All the fragments amplified from the urine samples were similar in size to that of BKV- WW. There was no size difference between strains of antigenic subtype I (which is described later) (from TI0 to D18-4 in Figure 4A), nor between strains of subtype I1 (D2 and B7 in Figure 4B), and none between the virus strains from different individuals (Figure 4A). The fragments of amplicons generated from the supernatant of cultures inoculated with urine samples also show various

Q.'"9'

sizes, ranging from 600-800 bp (see J7 and J8, subtype I in Fig 4A; JI and J3, subtype 11; J4 subtype 111 and J j and J2 subtype IV in Figure 48).

Similarly, size differences are seen in the PCR- generated DKA fragments from the supernatant of bacterial cultures containing cloned BKV plasmids. Com- pared with the well known strains DUN, IR, GS and WW (Figure 4A), the SB-like clones SB, IK, SB69, SB73, SB72-I, SB72-11, SB72-12, sB71- 6, SB71-7, SB71-9, and SB71-11 display a range of sizes (Figure 4B). Moreover, the fragments that were obtained from different clones of a single virus, e.g. the strains SB72, SB71 and DB (Figure 4B) also show a range of sizes. These variations in size seem unrelated to the genomic subtype differences in the late region for the same v i ru~es .~ '

GELOMIC FUNCTION OF BK\. 1709

Some of the recombinant genomes could not be amplified, e.g. SB69, SB73, SB72-1, DB-9 and others. This may be due to loss of primer annealing sites during viral propagation in cell culture. One of these clones, DB-9, has a 493-bp deletion in the NCR extending from the agnogene to the VP3 coding sequences. Such deletions may also explain why some of the clones are not productive after transfection into human embryo kidney cells.

The archetypal strain BKV-WW, which lacks positive active sequence etements reportedly will not grow in cultured cells," whereas strains BKV-PT and BKV-DUN, which contain duplicates of both the P50 and P68 fragments, grobv relatively well.21~27 This suggests that duplication of the block P favours viral growth in culture. The sequences of the blocks Q and R appear to be non-essential for growth in culture since strains PT and DUN have no intact blocks Q and R yet they grow as byell as strains that contain them (Figure 3)." There is no evidence to show that negatively acting elements are present in block R of BKV-WV"I~.~' Other investigators have reported e1,idence of tissue-specificity for some elements of the control region of the genome in different strains of BKV. This may explain the variable host range of BKV strains.93

In summary, variants of BKV can be demonstrated readily. The nucleotide sequences that constitute the NCR of some isolates are highly variable and this variation has been found to contribute to the \vide variety of Iytic and transforming phenotypes ijvhich are

I t is unlikely that rearrangements occurring during passage in culture represent the only mechanism for the formation of repeats in the NCR of BKV.

The relationship between differences in the NCR and the host ranges of BKV strains needs to be clarified. Clinical isolates of BKV may contain sub-populations of the virus ivhich have different control regions, permitting the \,irus population to adjust effectively to the range of transcription and replication factors a\,ailable in host cells.18 91 Rearrangements in the control region might affect tissue tropism and pathogenicity or might explain lvhy BKV, which is assumed to remain latent in the kidneys, can also be found in the tonsils,", in brain tumours and in pancreatic islet tumours.9'" There are a number of examples of how re-arrangements in the control region affect the tissue specificity, pathogenicity and oncogenicity of polyomaviruses18 'O," 93 However, the relationship between the variation in the control region of BKV and the capacity for transmission and reactivation is still unknown

evident for different s t r a i n S , i j , ~ ? . 1 8 , 3 8 . - 9 , 8 - , ~ l , ~ ~

Di,fere~ces in the 'em$' region The T-4g and t-Ag ir. different BKV strains are nearly identical. There are 9 aa differences between strains BKV-AS and BKV-DUX; the extreme carboxy terminal region of the 7-Ag of strain BKV-AS lacks 4 aa that are present in the corresponding region of strain BKV- DUh. l8 Strain BKV-IR, which has a deletion and an

insertion in the early region compared to BKV-PT, grows equally well in WI-38 cells.8q

Alteration of the analogous sequences of the T-Ag of SV4O leads to reduced virus yield and reduced propensity for growth in some cell lines," and may contribute to post-transcriptional regulation of the agnoprotein. I t is unclear if a comparable post-transcriptional mechanism operates in BKV isolates.

Alternfioizs in the late region Of seven BKV strains, MM, MT, DIK, WM', JL, DUK and AS, which have different control regions, the first five have been shown, by HAI, to be related antigenically to strain BKV-DUN, but not to strain AS. Among these six strains, 61 point mutations have been identified in the late coding region (2265 nucleotides). These variations are not randomly distributed, being present at the same position in different isolates. The reason for the distinct antigenicity of BKV-AS is intriguing; 108 point mutations have been detected in the structural proteins (VPI, VP2 and VP3) of BKV-AS when compared tvith BKV-DUN.Y8 It seems likely that the difference in antigenicity between these two viruses has its basis in the 32-aa differences in the viral late structural proteins, especially in a distinct cluster of 20-aa in the amino portion of VP1.18

To study the genetic basis of BKV strain antigenic heterogeneity, serological variants representative of the four BKV subtypes (PT, SB, AS, and IV. Table 1) were sequenced in the VPI region following PCR amplification." Each subtype group was found to have a characteristic nucleotide sequence. For the full sequence for VPI (326 amino acids), there were only 5 aa differences between the two serologically different sentinel strains SB (subtype 11) and AS (subtype 111). These were located in the aa 01-83 region. These results suggest that the subtype specific serological differences previously described for BKV isolates on the basis of HA1 tests are due to differences in the sequence of the aa 61-83 region of VPl."91

If these antigenic differences are stable and sufficiently distinct and reproducible, they will be valuable biological tools for use in molecular epidemiological investigations of BKV and in work aimed at understanding the mechanism of virus variation.

An antigen common to mouse polyomavirus strains has been localised to the amino terminal end of the major structural protein VPI, and is thought to be located internally in the intact virion. Possibly a similar common antigen exists in strains of BKV but is exposed on the surface of the virion. This could explain the broad reactivity of BKV strain SB with antisera to other BKV variants (Table 1). Howex-er, strain-specific serological responses seem to predominate as shobvn for isolates AS, IV and DB. Although all these viruses cross-react in HA1 tests with polyclonal antisera, the antibody titre to the homologous strain is the highest in every case.

Antibodies raised against BKV-PT cross-react weakly with BKV-SB, AS, IV and DB virions in HA1 tests.18~a1~9i Studies on the VPI of Py virus have shown that a single

210 LI TIN AND P. E. GIBSON

amino acid substitution (aa 92) can change biological characteristics such as plaque morphology, HA behaviour, the host range for tumorigenicity, viral replication and the ability of the virus to recognise cell surface

Therefore, it is possible that point receptors. mutations in the sequence may have partly removed and modified the dominant epitope(s) recognised on strain BKV-PT, but this needs to be clarified. In all variants of SV40, JCV and BKV the amino acid at position 92 of the VPI molecule is invariably alanine, which suggests that position 92 may be not as critical in these viruses as it is for polyomavirus.

Heterogeneity amongst viral genomes can arise in several ways: (I) natural genetic variation in the virus population resulting in the accumulation of point mutations in viz70;38,74,76 (2) the accumulation of variants during i n vitro propagation in cell c ~ l t u r e ~ ~ , ~ ~ (3) the use of different sources and methods for the derivation of molecular clones of BKV strains.”

The procedure for determining viral DNA sequences, which was developed for differentiating and subtyping the established strains of BKV, has been extended to clinical More than 100 individual strains, obtained from children, pregnant women, transplant recipients, HN-infected patients etc, including the strains BKV-IR (Italy) and BKV-DB (USA), were investigated by direct sequencing or restriction enzyme analysis of PCR product DNA containing the defined subtype-specific region (equivalent to nt 1744- 1812 of strain BKV- DUN).I5 All the strains could be assigned to one or other of the four genomic subtypes I (strain PT or GS), I1 (strain SB), 111 (strain AS) and IV (strain IV or DB) on the basis of their characteristic nucleotide sequence in the subtype- specific region. Dual infections with subtype I and one of the other subtypes was found, mainly in HIV-infected patients.11J92 These results attest to the stability of the subtype-specific region.

BKV subtype I was found most frequently in the study population, followed by subtypes 11, IV and 111. This order

101-103

of prevalence correlated well with that obtained by serological testing, although in some studies subtype I1 virus (strain SB, isolated in the UK) was not available for antigen p r o d ~ c t i o n . ~ ~ , ~ ~ Tavis et aL9’ found, in 993 blood samples collected in the USA from different age groups, that antibodies against the isolates BKV-PT (subtype I), BKV-AS (subtype 111) and BKV-DB (subtype IV), as detected by HAI, were widespread. Comparing one subtype with another, in the normal population from 1 to 80 years old, the highest percentage of antibody positive sera was recorded in tests against the BKV-PT virus. The prevalence of antibody against BKV-PT ranged between 65% and 80% whereas for strain BKV-DB the range was 47-64% and for strain BKV-AS it was 23-48%. Both the serological results and the genomic results strongly suggest that the four subtypes of BKV are independently circulating in the human population. Genetic subtyping methods have been shown to be reliable for estimating the prevalence of BKV infection in the population by screening urine or other samples. I 1,99,104

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to Dr J. C. Booth and Dr C. G. Teo for helpful discussions.

REFERENCES

Gardner, S. D., Field, A. M., Coleman, D. V. and Hulme, B (1971). New human papovavirus (BK) isolated from urine after renal transplantation. Lancet i, 1253-1257. Padgett, B. L., Walker, D. L., ZuRhein, G. M., Eckroade, R. J. and Dessel, B. H. (1971). Cultivation of papova-like virus from human brain with progressive multifocal leukoencephalopathy. Lancet i, 1257-1260.

GEKOMIC FUNCTIOK OF BKV

3. FI nner, F. (1976). The rules of nomenclature of vAA-uses Ii?iervirolog!i 7, 17-33.

4. Dougherty, R. M. and DiStefano, H. S. (1974). Isolation and characterization of a papovavirus from human urine. Proc. Soc. Exp. Biol. M e d . 146, 481- 187.

5. Penney, J. B. Jr and Narayan, 0. (1973). Studies of the antigenic relationships of the new humar. papovavirus by electron microscope agglutination. Infect. Irnmitn. 8, 299-300.

6. Padgett, B. L. and Walker, D. L. (1976). New human papovaviruses. Prog. Med. Virol. 22, 1-35.

7. Takemoto, K. K. and Mullarkey, M. F. (1973). Human papovaviruses, BK strain: Biological studies including antigenic relationships to simian virus 40. 1. Virol. 12, 625-6131,

8. Chesters, P. M., Heritage, J . and McCance, D. J , (1983). Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues. J Infect. Dis. 147, 676-684.

9. McCance, D. J. and Gardner, S. D. (1987). Papova- viruses: Papillomaviruses and polyomaviruses. In P(inciple5 and prnciire of clincnl virology, ed. by A. J. Zuckerman. J. E. Banatvala, and J. R. Pattison, pp. 479-506. John Wiley, Chichester.

10. Arthur, R. R. and Shah, K. V. (1989). Occurrence and significance of papovaviruses BK and JC in the urine. Prog. 'Wed. Virol. 36, 42-61.

11. Jin, L., Gibson, P. E., Booth, J. C. and Clewley, J . P. (1993) Genomic typing of BK virus in clinical specimens by direct sequencing of polymerase chain reaction products. 1, Med. Virol. 41, 11-17.

12. Jin, L., Pietropaolo, V., Booth, J. C., Ward, K. and Brown, D. W. (1995). Prevalence and subtype distribution of BK virus in healthy people and immunocompromised patients detected by PCR- restriction enzyme analysis. Cl in Ding. Viroi. 3,

13. Dynan, W. S. (1989). Modularity in promoters and enhancers. Cei! 38, 1-4.

14. Dhar, R., Seif, I. and Khoury, G. (1979). Nucleotide sequence of the BK virus DNA segment encoding small t antigen. Proc. Nnf l Acnd. Sci. USA 76,

15. Seif, I., Khoury, G. and Dhar, R. (1979). The genome of human papovavirus BKV. Cell 18, 963-933.

16. Frisque, R. J., Bream, G. L. and Cannella, M. 7. (1984). Human polyomavirus JC virus genome. 1. viroi. 51, 458-469.

17 . 'fang, R. C. A. and Wu, R. (1979). BK virus DNA: Complete nucleotide sequence of a human tumor virus. Science 206, 456-462.

18. Tavis, J. E., Walker, D. L., Gardner, S. D. and Frisque, R. J. (1989). Nucleotide sequence of the hcman polyomavirus A S virus, an antigenic variant of BK virus. 1. Virol. 63, 901-911.

19. Yoshiike, K. and Takemoto, K. K. (1986). Studies with BK virus and monkey lymphotropic papova-

285-295.

565-569.

virus. In, The papozmir idne , ed. by N. P. Salzman, pp. 295-326. Plenum Press, New York.

20. Walker, D. L. and Frisque, R. J. (1986). The biology and molecular biology of JC virus. In The pnpuva- uiridne (I), ed. by N. P. Salzman, pp, 327-381. Plenum Press, New York.

21. Seif, I., Khoury, G. and Dhar, R. (1979). BKV splice sequences based on analysis of preferred donor and acceptor sites. Nr&c Acid Res. 6, 3387-3398.

22. Frisque, R. J. (1983). Nucleotide sequence of the region encompassing the JC virus origin of DNA replication. 1. Vim/. 46, 170-176.

23. Del Vecchio, A. M., Steinman, R. A. and Ricciardi, R. P. (1989). An element of the BK virus enhancer required for DNA replication. 1. Virol. 63, 1514- 1524.

24. Ryder, K., DeLucia, A. L. and Tegtmeyer, P. (1983). Binding of SV40 A protein to the BK virus origin of DNA replication Virology 129, 239-245.

25. Deyerly, K. L. and Subramani, S. (1989). Human papovavirus BK early gene regulation in non- permissive cells. virology 169, 3855396.

26. Markowitz, R-B. and Dynan, W. S. (1988). Binding of cellular proteins to the regulatory region of BK virus DNA. 1. Viral. 6 2 , 3388-3398.

27. Markowitz, R-B., Tolbert, S. and Dynan, N. S. (1990). Promoter evolution in BK virus: Functional elements are created at sequence junctions. 1, Viuoi.

28. Deyerly, K. L. and Subramani, S. (1988). Linker scan analysis of the early regulatory region of human papovavirus BK. J Virol. 62, 3378--3387.

29. Kadonaga, J. T., Jones, K. A. and Tjia, R. (1986). Promoter- specific activation of R L 4 polymerase II transcription by Spl . Trends Bicchem. Sci. 11,

30. Briggs, M. R., Kadonaga, J. T., Bell, S. P. and Tjian, R. (1986). Purification and biochemical characterization of the promoter specific transcription factor, Sp;. Science 234, 47-52.

31. Lee, W., Haslinger, A,, Karin, M. and Tjian, R. (1987). Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV4O. ,Yature 325,

32. Deyerle, K. L., Cassill, J. A. and Subramani, S. (1987). Analysis of the early regulatory region of the human papovavirus BK. Virology 158, 181-193.

33. Ayer, D. and Dynan, W . S. (1988). Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences. A.4~l. Cell. Biul. 8,

34, Bolen, J . B. and Consigli, R. A. (1980). Separation of neutralizing and hemagglutination -inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate- derived polypeptides of polyoma virus. 1. Viral. 34, 119-129.

35. Anders, D. G. and Consigli, R. A. (1983). Chemical cleavage of polyomavirus major structural protein

64, 2411-2415.

20-23.

368-372.

202 1-2033.


36.

3 7.

38.

3 9.

40.

41

42

43

44.

45

46

47

48

49.

50.

31.

VPI: Identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region. 1, Virol. 48, 197-205 Lee, W., Mitchell, P. and Tjian, R. (1987). Purified transcription factor AP-1 interacts with TPA- inducible enhancer elements. Cell 49, 741-752. Sugimoto, C., Harz, K., Taguchi, F. and Yogo, Y. (1989). Growth efficiency of naturally occurring BK virus variants in vivo and in vitro. 1. Virol. 63,

Rubinstein, R. and Harley, E. H. (1989). BK virus DNA cloned directly from human urine confirms an archetypal structure for the transcription control region. Virus Genes 2, 157-165. Cassili, J. A., Deyer!e, K. and Subramani, S. (1989). Unidirectional detection and linker scan analysis of the late promoter of the human papovirus BK. Virology 169, 172-189. Cassill, J . A. and Subramani, S. (1989). Naturally occurring deletion in the enhancer repeats of the human papovavirus BK optimizes early enhancer function at the expense of late promoter activity. Vivobgy 170, 296-298. Yang, R. C. A. and Wu, R. (1979). Comparative study of papovavirus DNA: BKV (MM), BKV (WT) and SV40. Kucleic. Acids Res. 7, 651-668. Yang, R. C. A. and Wu, R. (1979). BK virus DNA sequence coding for the amino-terminus of the T-antigen. Virology 92, 340-352. Yang, R. C. A. and Wu, R. (1980). BK virus DKA sequence coding for the t and the T antigens and evaluation of methods for determining sequence homology. 1. Virol, 34, 416-430. Seif, I . and Khoury, G. (1979). Nucleotide sequence of the BK virus DNA segment encoding small t- antigen. Proc. X a f l . h a d . Sci, USA 76, 565-569. Cassil, J . A. and Subramani, S. (1988). The late promoter of the human papovavirus BK is contained within early promoter enhancer region. Virojogy 166, 175-1 85. Portolani, M., Barbanti-Brodano, G. and LaPlaca, M. (1975). Malignant transformation of hamster kidney cells by BK virus 1. Vivo2. 15, 420-422. Purchio, A. F. and Fareed, G. C. (1979). Transfor- mation of human embryonic kidney cells by human papovavirus BK. J, Vivol. 29, 763-769. Shah, K. V., Daniel, R. W . and Strandberg, J. D. (1973). Sarcoma in a hamster inocuiated with BK virus, a human papovavirus. 1, Nafl Cancer Ins/ . 54, 945-950. Costa, J . , Yee, C., Tralka, T. S. and Rabson, A. S. (1976). Hamster ependymomas produced by intra- cerebral inoculation of a human papovavirus (MMV). 1. h'atl Cancer. Inst. 56, 863-864. Takemoto, K. K., Linke, H., Miyamura, T. and Fareed, G . (1. (19793. Persistent BK papovavirus: infection of transformed human fetal brain cells.

Pater, A. and Pater, M. M. (1986). Transformation

3 195-3 199.

I. Vii'Gl. 29, 1177-1185.

independence by combination of BK virus DKA and the harvey-ras oncogene. 1. Vivol. 58, 680-683.

52. Nakshatri, H., Pater, M. M. and Pater, A. (1988). Functional Role of BK virus tumor antigens in transformation. J Virol. 62, 4613-4621.

53. Oldstone, M. B. A. (1991). Molecular anatomy of viral persistence. I. i/ivoi. 63, 6381-6356.

54. Kalderon, D., Richardson, W. D., Markham, A. F. and Smith, A. E. (1984). Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311, 33-38.

55. Clever, J. and Kasamatsu, H. (1991). Simian virus 40 VP2/3 small structural proteins harbor their own nuclear transport signal. Virology 1813, 78-90

56. DePamphilis, M. L. and Bradley, M. K. (1986). Replication of sV40 and polyoma virus chromo- somes. In, The Papovaviridae, I1 ed. by N. P. Salzman, pp. 99-140. Plenum Press, New York.

57. Dixon, K., Ryder, B-J. and Burch-Jaffe, E. (1982). Enhanced metastasis of tumors induced by a sV40 small T deletion mutant, Nature 296, 672-675.

58. Mathews, B. J., Levine, A. S. and Dixon, K. (1987). Deletion mutations ir, the small t-antigen gene alter the tissue specificity of tumors induced by simian virus 40. 1. Virol. 61, 1282-1285.

59. Shah, K. V., Ozer, H. L., Ghazey, H. N. and Kelly, T. J. Jr. (1977). Common structural antigen of papovaviruses of the simian virus 40- polyoma subgroup. 1, Virol. 21, 179-186.

60. Bolen, J . B., Anders, D. G., Trempy, J . and Consigli, R. A. (1981). Differences in the subpopulations of the structural proteins of polyoma virions and capsids: Biological functions of the multiple of VPI. 1. Viral. 37, 80-91.

61. Brady, J. N. and Salzman, N. P. (1986). The papovaviruses, general properties of pol yoma and SV40. In The Papovaviridae, I, ed. by h'. P. Salzman, pp. 1-26. Plenum Press, Kew York.

02. Wychowski, C., Benichou, D. and Girard, M. (1986). A domain of SV 40 polypeptide VPI tha t specifies migration into the cell nucleus. EMBO 1, 5, 2569-25 76.

63. Chang, D., Haynes 11, J. L., Brady, J. N. and Consigli, R. A. (1992). The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP;. V:'rdoKd 189, 821-827.

64. Moreland, R. B., Montross, L. and Garcea, R. L. (1991). Characterization of the DNA-binding properties of the polyomavirus capsid protein VPI . 1, Virol. 65, 1168-1176.

65. Montross, L., Watkings, S., Moreland, R., Mamon, H., Caspar, D. D., and Garcea, R. L. (1991). Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein \'PI. J Vi '.

66. Streuli, C. H. and Griffin, B. E. (1987). Myristic acid is coupled to a structural protein of polyoma virus

65, 4991-4998.

of human embryonic kidney cell to anchorage and SV40. Nafure 326, 619-622. .

~ GENOMIC FUSCTION OF BKV 7 1 3

67.

68.

69.

70.

71.

72.

73.

74.

75.

70.

77.

78.

79.

80.

81.

82.

Krauzewicz, N., Streuli, C. H., Stuart-Smith, N., Jones, M. D., Wallace, S. and Griffin, B. E. (1990). Myristylated polyomavirus VP2: role in the life cycle of the virus. J . Viuol. 64, 4414-4420. Wychowski, C., Benichou, D. and Girard, h4, (1987). The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence. J Viroi. 61, 3862-3869. Gharakhanian, E., Takahashi, J . and Kasamatsu, H. (1987). The carboxyl 35 amino acid of SV3O VP3 are essential for its nuclear accumulation. Virulogy

Dingivall, C. and Laskey, R. A. (1986). Protein import into the cell nucleus. Aiznii. Reo. Cell B i d 2,

Ng, S. C., Mertz, J . E., Sanden-Will, S. and Bina, M. (1985). Simian virus 40 maturation in cells harbor- ing mutants deleted in the agnogene. ]. Biol. Chem.

Barkan, A. and Mertz, J . E. (1981). DNA sequence analysis of simian virus 40 mutants with deletions mapping in the leader region of the late mRNA’s : mutants with deletions similar in size and position exhibit varied phenotypes. 1. Vim/, 3 7 , 730-73 7 . Carsbvell, S. and Ahvine, J. C. (1986). Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VPI, the major capsid protein. J Viuoi.

Sundsfjord, A,, Johansen, T., Flaegstad, T. et iii.

(1990). At least two types of control regions can be found among nuturally occurring BK virus strains.

Howley, P. M., Mullarkey, h4. F., Takemoto, K. K. and Martin, M. A. (1975). Characterization of human papovavirus BK DNA. J Viuol. 1 5 , 173-181. Martin, J. D., Padgett, B. L. and Walker, D. L. (198.3). Characterization of tissue culture induced heterogeneity in DNAs of independent isolates of JC virus. 1. Gen. Virol. 64, 2271-2280 van der Noordaa, J. (1976). Infectivity, oncogenicity and transforming ability of BK virus and BL 1,irus DLA. J Gem Viuo/. 30, 371-373. Watanabe, S., Soeda, E., Uchida, S. and Yoshiike, K. (1984). DNA rearrangement affecting expression of the BK virus transforming gene. ]. Visol. 51, 1-6. Pater, A,, Pater, M . M., Chang, L. S., Slawin, K. and di Mayorca, G. (1983). Multiple origins of the coniplementary defective genomes of RF and origin proximal sequences of GS, two human papovavirus isolates. Virology 131, 426-436. Pater, M. M., Pater, A. and diMayorca, G. (1981). Genome analysis of M G virus, a human papovavirus. 1. Viuol. 39, 968-972. Knowles, W. A,, Gibson, P. E. and Gardner, S. D. (1989). Serological typing scheme for BK-like isolates of human polyoma\-irus. 1, kled. L’iroi. 28,

Yoshiike, K, Miyamura, 7, Chan, H. W. and Takemoto, K. K. (1982). Two defective DNAs of human polyomavirus JC adapted to grovvth in

15 7, 440-348.

3 6 7-3 90.

260, 1127-1132.

60, 105551061.

1. lf’ir01. 64, 3863-3871.

118-123.

human embryonic kidney celis. 1. Vim/. 42, 395- 401

83. Melv, R. T., Lecatsas, G., Prozesky, 0. W. and Harley, E. H. (1981). Characteristics of BK papoira- virus DNA prepared directly from human urine. bztervirology. 16, 13-19,

83. Howley, P. M., Khoury, G., Byrne, J . C., Takemoto, K. K. and Martin, M. A. (1975). Physical map of the BK virus genome. 1, Viral, 16, 959-973.

83. Chauhan, S., Lecatsas, G. and Harley, E. H. (1984). Genome analysis of BK (WW) viral DNA cloned directly from human urine. InfeuzJiralogy 22, 170- 176.

84. Yang, R. C. A. and Wu, R. (1978). BK virus DNA: Cleavage map and sequence analysis (tumor virus/ simian virus 40/leader mRNA sequence). Proc. Natl. Acad. Sci. USA 75, 2150-2154.

85. Rubinstein, R., Pare, N. and Harle),, E. H. (1987). Structure and function of the transcriptional control region of nonpassaged BK virus. 1. l&ol. 61,

86. Gibson, P. E. and Gardner, S.D. (1983). Strain differences and some serological observations on several isolates of human polomavimses. In, Po/!inniiiviuitses und Hztvnan xtI.iero/ogicn/ Diseiists, ed. by J. L. Sever and D. L. Madden, pp. 119-132. Alan R. Liss, New York.

87. Pagnani, M., Negrini, M., Reschiglian, P. et ui. (19861. Molecular and biological properties of BK virus-IR, a BK virus variant isolated from a human tumor. Virobgy 59, 500-505.

88. Frisque, R. J , , Martin, J . D., Padgett, B. and W-alker, D. L. (1979). Infectility of the D h A from four isolates of JC virus. J Virol. 32, 476-482.

89. Gheysen, D., van de Voorde, A., Contreras, R., Vanderheyden, J.! Duerinek, F. and Fiers, W. (1983). Simian XTirus 40 mutants carrying extensive deletions in the 72-base- pair repeat region. 1, Vivol. 47, 1-14.

90. Flaegstad, T., Sundsfjord, A,, Arthur, R. R., Pedersen, M., Traavik, T. and Subramani, S. (1991). Amplification and sequencing of the control regions of BK and JC virus from human urine by polyomer- ase chain reaction. Virology 180, 553-560.

91. Tavis, J. E., Frisque, R. J. . Walker, D. L. and White 111, F. A. (1990). Antigenic and transforming properties of the DB strain of the human polyomavirus BK virus. Virology 178. 568-5 72.

92. Jin, L. (1993). Antigenic Vuriiiizts I J ~ Hitnim Po/yomii- ilirtts / B K V ) ; DKA Seqrteiice A~niysis of n Regioti of t he

Pwfeiir VPI zol!icl: rontsiinc Q Sr €p i tope , Thesis submitted to the University of London for the degree of PhD.

93. Grinnell, B. W., Berg, D. T. and Walls, J. D. (1988). Negative regulation of the human polyomavirus BK enhancer involves cell-specific interaction with a nuclear repressor. Mol. Cell Biol. 8, 348-3457,

94, Rubinstein, R., Schoonakker, C. A. and Harley, E. H. (799:). Recurring theme of change in the

1747-1750.


95.

96.

97.

98.

99.

transcriptional control region of BK virus during adaptation to cell culture. 1. Virol. 6 5 , 1600-1604. Goudsmit, J., Wetheim-van Dillen, P., Sterien, A. V. and Vander Noordad, J. (1982). The role of BK virus in acute respiratory tract disease and the presence of BKV DNA in tonsils. 1. Med. Virol. 10, 91-99. Corallini, A., Pagnani, M., Viadana, P. et al. (1987). Association of BK virus with human brain tumors and tumors of pancreatic islets. Int. 1. Cancer. 39,

Cole, C. N. and Stacy, T. P. (1987). Biological properties of simian virus 40 host range mutants lacking the COOH-terminus of large T antigen.

Sugimoto, C., Hara, K., Taguchi, F. and Yogo, Y. (1 990). Regulatory DNA sequence conserved in the course of BK virus evolution. 1. Mol. Evolut. 31,

Jin, L., Gibson, P. E., Knowles, W. A. and Clewley, 1. P. (1993). BK virus antigenic variants: Sequence analysis within the capsid VPI epitope. 1. Med. Virol. 39, 50-56.

60-67.

Vi~010gy 161, 170-180.

485-492.

100. Shah, K. V., Daniel, R. W. and Kelly, T. J. Jr. (1977). Immunological relatedness of papovaviruses of simian virus 40-polyoma subgroup. Infect. Imrnun.

101. Freund, R., Calderone, A., Dawe, C. J. and Benjamin, T. L. (1991). Polyomavirus tumor induction in mice: Effects of polymorphisms of VPI and large T antigens. 1. Viral. 6 5 , 335-341.

102. Freund, R., Garcea, R. L., Sahli, R. and Benjamin, T. (1991). A single-amino acid substitution in polyomavirus VP1 correlates with plaque size and haemagglutination behaviour. 1. Virvl. 6 5 , 350- 355.

103. Dubensky, T. W., Freund, R., Dawe, C. J. and Benjamin, T. L. (1991). Polyomavirus replication in mice: Influences of VPI type and route of inoculation. 1. Virol. 6 5 , 342-349.

104. Jin, L. (1993). Rapid genomic typing of BK virus directly from clinical specimens. Molecular and Cellular Prvbes. 7, 33 1-334.

18, 558-560.

Documents

Genomic Function and Variation of Human Polyomavirus BK (BKV)