© 1998 by Oxford University Press
Journal of the National Cancer Institute Monographs, No. 23, 79-88,
1998
© 1998 Oxford University Press
Novel Organizational Features, Captured Cellular Genes, and Strain Variability Within the Genome of KSHV/HHV8
* Affiliations of authors: J. Nicholas, J.-C. Zong, D. J. Alcendor, D. M. Ciufo, L. J. Poole, R. T. Sarisky, C.-J.- Chiou, X. Zhang, X. Wan, G. S. Hayward, Molecular Virology Laboratories, Oncology Center, The Johns Hopkins School of Medicine, Baltimore, MD; H.-G. Guo, M. S. Reitz, Institute of Human Virology, University of Maryland, Baltimore.
Correspondence to: Gary S. Hayward, Ph.D., Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, 725 N. Wolfe St., WBSB 317, Baltimore, MD 21205. E-mail: Gary.Hayward{at}qmail.bs.jhu.edu
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Abstract |
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Strong serologic and molecular probe correlations indicate that the newly discovered gamma herpesvirus KSHV or HHV8 is the likely etiologic agent of all forms of Kaposi's sarcoma as well as BCBL/PEL and MCD in patients with acquired immunodeficiency syndrome (AIDS). Two large segments of HHV8 DNA from an AIDS-associated BCBL tumor covering genomic positions 0-52 kilobase [kb] and 108-140 kb have been cloned, mapped, and partially sequenced. Our studies have focused on novel viral proteins encoded within a 13-kb divergent locus (DL-B) by nine captured homologues of cellular genes, including vIL-6, vDHFR, vTS, vBcl-2, three C-C beta chemokines (vMIP-1A, vMIP-1B, and vBCK), and two LAP/PHD subclass zinc finger proteins (IE1A and IE1B). The HHV-8 vIL-6, vDHFR, vTS, and vBcl-2 proteins have all been shown to be active in a variety of appropriate functional assays, and transcripts from vIL-6, vMIP-1B, vIE1-A, vIE1-B, and vDHFR genes are all expressed as abundant single messenger RNA species after butyrate or phorbol ester (TPA) induction of the lytic cycle in HHV8-positive BCBL cell lines. All of these genes lie within a divergent transcriptional domain that contains a single central enhancer and associated untranslated leader region plus seven distinct proximal promoters, some of which are negatively regulated through AP-1 and ZRE motifs by the EBV ZTA transactivator. This region also encompasses a predicted complex oriLyt domain of 1050 bp that is duplicated in inverted orientation adjacent to the T0.7 latency RNA in another large divergent locus (DL-E). We have previously described three distinct subtypes of the HHV8 genome that differ by 1.0%-1.5% at the nucleotide level within the ORF26 and ORF75 genes. Certain strains or clades appear to have preferential geographic distributions, but it is not known as yet whether there are any specific disease associations. Interestingly, the A, B, and C subtypes of HHV-8 also proved to differ dramatically in coding content at both the extreme left and right ends of the unique segment of the genome as well as in the positions of the junctions with the terminal repeats. On the left-hand side, the receptor-like ORF-K1 protein is highly variable with A-strain subtypes displaying 15% amino acid differences from C strains and up to 30% differences from B strains. On the right-hand side, two unrelated alternative types of the putative multiple membrane spanning ORF-K15 protein are found.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion and ConclusionsReferences |
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Genome Characteristics of Gamma Herpesviruses
The complete genomic DNA sequences of five distinct gamma
herpesviruses are now available. A comparison of the organization
and gene content of Epstein-Barr virus (EBV) (1), herpesvirus saimiri (HVS) (2), EHV-2 (3), HHV-8 (4), and MHV68 (5)
is shown in Fig. 1.
Major blocks (I, II,
III, and IV) of conserved genes present in all herpesviruses are
shown and individual conserved genes that have residual homology
among all of the five gamma herpesvirus genomes are given as
solid bars. Hatched or open bars show those genes that are
present in only a subset of these viruses or are unique to
individual viruses. These genes tend to group within six regions
referred to as divergent loci (DL) A, B, C, D, E, and F. Prior to
the discovery of HHV-8 (6), the human
B-cell trophic virus EBV and the squirrel monkey T-cell
lymphoma-associated virus HVS were designated as the prototypes
of two subgroups of gamma herpesviruses, namely, 
1 and
2. A number of old-world primate viruses similar to EBV are
also clearly 1 viruses and several other new-world primate
2 viruses (also known as Rhadinoviruses) are closely related
to HVS. More recently, several equine (EHV-2), bovine (BHV-4,
AHV-1), and murine (MHV68) viruses have also been tentatively
assigned to the 2 subclass based on data about their gene
content and genome organization, although a case could be made
for assigning EHV-2 to a separate subclass because of its higher
GC content and the presence of large direct terminal repeats
rather than the otherwise typical multicopy short terminal
repeats (3).
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),
and ori-P (
) in EBV and the duplicated proposed ori-(L) and
ori-(R) domains in HHV-8 (The genome organization of EBV differs from that of HVS primarily by the presence of the unique B-cell latency genes and other features, including ori-P, EBNA-1, EBNA-2, EBNA-3abc, LPNA, EBERs, and LMP-1 and 2 as well as the B-cell receptor glycoprotein, the lytic cycle triggering DNA-binding bZip protein ZTA, the large (10 x 3.1 kilobase [kb]) internal IR repeats, and the duplicated ori-Lyt domains (DS-L and DS-R). In their place, HVS has genes encoding the TIP, STP, and vDHFR proteins together with the HSUR small RNAs at the left-hand end (DL-A region) and the vTS(ORF70), vFLIP(ORF71), vCycD(ORF72), ORF73, and an IL8R-like vGCR (ORF74) gene in DL-E. HHV-8 is clearly not a
1 virus and it has some features that closely
resemble HVS, such as the vFLIP (K13) (7),
vCycD, LANA(ORF73), and vGCR gene block in DL-E (8), and the presence of short multicopy terminal
tandem repeats, but it also differs significantly from HVS by its
higher overall GC content (55%), lack of CpG-suppression,
insertion of the IRF gene block (DL-D3), and major additions or
alterations in several of the divergent loci, especially in DL-A,
DL-B, and DL-F. At the present time, equally compelling arguments
can be made either for classifying HHV-8 along with all of the
other non-EBV-like gamma herpesviruses together in a super 2
subgroup or for giving both HHV-8 and EHV-2 separate status as
3 and 4 viruses. Alternatively, it might be wise to
abandon any attempt at rational subdivisions within the gamma
herpesviruses. Novel Features of KSHV/HHV-8
Major features of the HHV-8 genome were described previously by Moore et al. (9), Cesarman et al. (8), Russo et al. (4), and Nicholas et al. (10). These include the lack of all EBV-specific features and the absence of most HVS specific features, except for the presence of vTS and vDHFR genes (both at new locations) and retention of the vFLIP, vCyc-D, LANA, and vGCR block. Several vIRF-like genes (e.g., ORF-K9) (11) and other potential novel genes (ORF-K8, K8.1, and K9-K11) lie in the DL-D2 and D3 regions, and a vOX-2 homologue (ORF-K14) has been inserted adjacent to the vIL8R-like vGCR gene in DL-E. A series of novel viral-encoded cytokine genes map within DL-B e.g., vIL-6 (K2), vMIP-1A (MIP-I, K6), vMIP-1B (MIP-II, K4), and vBCK (K4.1), together with vTS, vDHFR, and two LAP/PHD class zinc finger protein genes referred to as IE1-A (K5) and IE1-B (K3) (10-13). Two novel abundant short RNAs (T1.1/Nut/K7 and T0.7/K12), which are unlike the HSURs of HVS or the EBERs of EBV, have been described (14,15) that also map within DL-B and DL-E, respectively. Finally, two small novel open reading frames (ORFs), K1 and K15, occur at the left and right ends of the HHV-8 genome (DL-A and DL-F) and are positional equivalents of the latency and transformation-related LMP-1 and LMP-2 membrane glycoprotein genes of EBV and the strain variable TIP and STP genes of HVS (16,17).
Many of these genes appear to be likely to contribute to the unique biology of HHV-8, but evidence for any direct involvement in disease processes must await extensive analysis of the patterns of gene expression in different cell types in Kaposi's sarcoma (KS) and other tumors, etc., and detailed examination of the functions of their protein products.
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Materials and Methods |
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The sources of the body cavity-based lymphoma (BCBL) and KS DNA samples and cell lines used have all been described previously, as well as the procedures used for library preparation, subcloning, and genomic or PCR sequencing of HHV-8 DNA (10,13,18-20). Procedures used for transcriptional and functional analysis of vIL-6 were also described previously (13). Details of the functional analysis of vTS and vDHFR, the transcriptional evaluation of the DL-B region, and the sequence data for the LHS and RHS ends of HHV-8(BCBL-R) will be presented elsewhere.
New HHV-8 phage lambda clones not previously described include
D-S1 (-1.8/14.5-kb), E-A2 (108.3/120.5-kb), and
E-C2 (115.7/129.0-kb), all in the DASHII background,
and B3-2 (120.6/137.4) in the EMBL3 background (Fig. 2,
A). These were all isolated from either of
two phage libraries generated from size selected Sau3A partial
digests of a BCBL tumor DNA sample described previously that we
refer to as BCBL-R (10,19). D-S1 was
identified by hybridization to a probe representing the
right-hand terminus of D3-80(ORF6), E-A2 by
hybridization to a T0.7 gene probe generated by polymerase chain
reaction (PCR) based on sequence data from Zhong et al. (14), E-C2 by hybridization to a probe
generated from the right-hand terminus of B6-1(ORF71), and
B3-2 by hybridization to a probe representing the left-hand
terminus of B6-1 (ORF75). The map coordinates of those and
our previously described BCBL-R phage lambda clones (10,19) were confirmed by restriction enzyme cleavage
mapping and terminal sequencing to correspond to genome positions
in the HHV8(BC-1) genomic DNA sequence of Russo et al. (4) as defined in parenthesis as follows:
D-VR3A (14.7/26.7 kb) and D-VR4A (18.5/31.2 kb) in the
DASHII background and D3-80 (5.4/22.9 kb), C7-1
(19.3/37.9 kb), C2-1 (27.8/47.2 kb), A12-1 (35/51 kb),
and B6-1 (121.7/134.4-kb), all in the EMBL3 background.
New plasmid subclones used for additional sequencing included
pDJA60 (BamHI/EcoRI 2.9 kb encompassing ori-L),
pDJA61 (BamHI/BamHI, 3.5 kb encompassing the
LHS TTR, ORF-K1A, and part of ORF4), pDJA62 (HindIII/SalI, 3.3 kb encompassing ORF-K15A and the RHS TTR),
plus pDJA63 (EcoRI/EcoRI, 6.0 kb encompassing
T0.7 and ori-R).
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Extension of the BCBL-R Phage Library
We have described previously the identification and sequencing of
nine novel captured cellular genes mapping between ORF11 and
ORF17 within DL-B on the LHS of the HHV-8(BCBL-R) genome, as well
as vCycD (ORF72) and an IL8R-like vGCR(ORF74) within DL-E on the
RHS (10,13,19-21). In the course of that
work, two sets of contiguous phage lambda clones encompassing
ORF6 to ORF31 (now defined as map coordinates 5.4-51 kb) and
ORF72 to ORF75 (map coordinates 120.6-134.4 kb) were assembled.
These two blocks have subsequently been extended both internally
between ORF64 and ORF72 to completely cover the remainder of the
DL-E region as well as toward the ends of the genome to encompass
DL-A at the extreme LHS boundary and DL-F at the extreme RHS
boundary, including proximal copies of the terminal tandem
repeats (TTR). New phage clones D-S1, E-A2, E-C2,
and B3-2 were identified by plaque hybridization with probes
representing the left-hand side (LHS) end of D3-80 (10), the T0.7 transcript (14), and the right-hand side (RHS) end of B6-1 (19), and short regions at the termini of
each insert were sequenced. A summary of the map locations of a
total of seven characterized phage lambda clones covering 53 kb
at the left end (positions -1.8 to 51 kb), and four other
selected lambda clones covering 30 kb at the right end (positions
108 to 138 kb) of the HHV-8(BCBL-R) genome are shown in Fig. 2
, A.
An Inverted Duplication Contains Potential Origin Features
Our analysis has revealed the presence of a 1050-bp domain in
DL-B between ORF-K4.2 and IE1-A (K5) that has typical features of
a herpesvirus lytic cycle DNA replication origin, including
clustered consensus binding motifs for AP-1 (JUN/FOS) and CTF,
multiple short repetitive motifs containing XcaI and PvuII sites that resemble features of EBV ori-Lyt, two FspI/SphI motifs matching sites found within
CMV ori-Lyt, and two long AT-rich palindromes, including
ATATATATATATATATAT, which resemble the core loop structures of
HSV ori-L and ori-S and are also found in the HHV6 and HHV7 lytic
origins. Most remarkably, the entire 1050 bp is duplicated as an
almost identical inverted copy between T0.7 (K12) and vFLIP
(ORF71) toward the other end of the genome within DL-E. Notably,
the positions of the two putative ori-Lyt domains in HHV-8
closely match those of the two directly repeated 800-bp ori-Lyt
domains in EBV and, as in EBV, both also lie directly adjacent to
high GC-content tandem repeats. In HHV-8, these repeats are
represented as both 20-bp and 30-bp tandem arrays in the LHS
region (DL-B) and as two types of 23-bp tandem repeats in the RHS
region (DL-E), and they are positionally analogous to the 102-bp PstI and 125-bp NotI tandem repeats in EBV,
respectively. Interestingly, MHV68 also has equivalent sets of
40-bp and 100-bp GC-rich tandem repeats at these locations, and
EHV-2 has a total of four sets of both direct and inverted
duplicated palindromic regions with (AT)n core loops
and multiple AP-1 binding sites on the stems within or adjacent
to the DL-B and DL-E regions. Only HVS appears to lack these
features. The locations of the predicted LHS ori-Lyt domains
within DL-B(ori-L) and DL-E (ori-R) are shown in Fig. 2, B.
Evaluation of Transcriptional Control Elements
We recognize eight distinct promoter domains within the 13-kb
DL-B region, including a 500-bp divergent promoter/enhancer (ENH)
between vMIP-1A and T1.1 that functions as a relatively weak
constitutive plus butyrate and TPA-inducible promoter in both
orientations in transient reporter gene assays in Vero, HeLa, and
U937 cells. This domain contains a predicted rightwards noncoding
leader exon of 45 bp that together with its upstream TATA-box
region is duplicated in a partially conserved manner within the
two proposed ori-Lyt domains. In fact, within the DL-B region,
the arrangement of the leftwards-oriented leader (ENH version)
together with the vMIP-1A and IE1-A genes, followed by the leader
(ori-L version) plus vMIP-1B and IE1-B genes in the same
orientation, suggests another ancient duplication that may
perhaps have occurred prior to the acquisition of the other
inserted cellular complementary DNAs (cDNAs) in DL-B (Fig. 3). Note that the 13-kb DL-E domain also
appears to represent a divergent transcriptional unit with a
single potential ENH-like domain associated with several
recognizable transcription motifs between ORF73 (LANA) and
ORF-K14 (vOX-2), but no functional data are available here as
yet.
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Curiously, both orientations of the divergent ENH-like control domains in DL-B and DL-E contain consensus 7-bp ZRE binding motifs (22,23) either very close to or slightly downstream from the TATA motifs, and both orientations of the DL-B ENH proved to be down-regulated by cotransfection with the EBV ZTA transactivator in Vero cells (Chiou C-J, Hayward GS: data not shown). Many of the proximal promoters also contain adjacent ZRE motifs either upstream or downstream of their TATA motifs, and several of the potential ZREs in DL-B are also classical JUN/FOS binding AP-1 sites. These findings raise the possibility that some key HHV-8 lytic cycle promoters may be regulated negatively by EBV in dually infected BCBL cell lines and tumors, and they also reinitiate old unsolved questions about whether there may be a cellular homologue of the EBV lytic cycle triggering protein ZTA, which HHV-8 may use.
Expression of messenger RNAs (mRNAs) from most of the genes in
DL-B (e.g., vDHFR, vTS, vMIP-1B, IE1-B, IE1-A, and T1.1) has been
found to occur only after butyrate induction of the HHV-8 lytic
cycle in the HBL-6/BC-1 (EBV-positive) cell line and after either
butyrate or TPA induction in the BCBL-1 (EBV-negative) cell line.
In contrast to TPA induction, butyrate induction appears to be
abortive (to the extent that the viral mRNAs are induced only
transiently and disappear after between 24 and 48 hours), and
curiously TPA does not induce the lytic cycle genes efficiently
in the dual EBV- and HHV-8-infected HBL-6 cell line (10,24,25). vIL-6 gives low-level constitutive
expression in the HBL6/BC1 cell line but is also greatly
increased by induction (11,13). Both the
predominant vIL-6 (1.0-kb) and vMIP-1B (0.8-kb) mRNAs produced
after induction have been found by primer extension analysis to
represent short monocistronic unspliced mRNA species (Poole L,
Ciufo D, Hayward GS: data not shown). However, both the IE1-A and
IE1-B genes, which lack identifiable proximal promoters, contain
excellent splice-acceptor motifs, and the vIL-6 and vMIP-1A genes
also appear to have the potential to occur as either spliced or
unspliced versions (Fig. 3).
Functional Activity of the Viral IL-6, Dihydrofolate Reductase (DHFR), and Thymidylate Synthetase (TS) Genes
Considering that the multistep captures of those cellular cDNAs are likely to have been relatively ancient evolutionary events, most such genes that are retained are expected to be functionally active, although they may have altered their functions in subtle ways either by broadening the substrate or target specificity or by acquiring constitutive rather than conditional activity that may lead to interference with normal host mechanisms or apoptotic or immune responses, etc. We have previously shown that the HHV-8-encoded vIL-6 produced from an SV2-vIL-6 plasmid in transfected rat embryo fibroblasts substitutes for human IL-6 in promoting growth of B9 mouse myeloma cells in a manner that uses the interleukin 6 (IL-6) receptor (13). It also mimics human IL-6 by inducing specific mRNA and DNA-binding transcription factors associated with the acute-phase response in HEP-3B cells [(13); Wan X.-Y., Nicholas J: manuscript in preparation)]. Some properties and functions of the HHV-8-encoded Cyc-D (ORF72), the IL-8R-like vGCR (ORF74), vMIP-1A (K6), and vIL-6 (K2) from HHV-8(BC1) have also been described by others (8,11,26,27).
We have also more recently cloned the HHV-8-encoded vDHFR and vTS genes into bacterial glutathione S-transferase (GST) and mammalian Flag-tagged vector systems (Sarisky R, Ciufo D, Zhang X, Chiou C.-J, Hayward GS: manuscript in preparation). Stable DHFR-negative CHO cell lines constitutively expressing the tagged vDHFR have been generated by Neo co-selection and these proved to be susceptible to cell killing with either 10 or 100 mM methotrexate. Furthermore, a methotrexate-resistant high copy number subclone efficiently bound to a fluorescent tagged dihydrofolate substrate as assayed by fluorescence-activated cell sorter analysis. Similarly, a transfected GST/vTS fusion protein plasmid proved both to rescue a thymidylate synthetase-negative Escherichia coli strain in Thy- medium and to make the host cells susceptible to killing by fluorouracil. All of these properties indicate that the vIL-6, vDHFR, and vTS genes retain the normal functional activities of their cellular homologues, although we have yet to discover whether or not their substrate or target specificities have been altered. Evidence that the HHV-8 encoded vBcl-2 (ORF16) protein functions similarly to human Bcl-2 to block apoptosis has also been presented (20,28).
Terminal Heterogeneity on the Right-Hand Side
Selective sequencing of relevant terminal segments of
HHV-8(BCBL-R) DNA subcloned into plasmids from the phage lambda
clones D-S1, E-A2, and B3-2 has also been carried
out (Alcendor D, Zhong J.-C, Wan X, Nicholas J, Guo H, Reitz M,
et al: manuscript in preparation). At the extreme RHS end of the
unique segment of the HHV-8(BCBL-R) genome a 3.3-kb HindIII to SalI insert from B3-2 subcloned in plasmid
pDJA62 was completely sequenced by standard M13 shotgun
procedures to extend our previous UPS75 DNA sequence (Genbank
accession no U85269). The results revealed a match to the
published HHV-8(BC-1) sequence across the N-terminus of ORF75,
but with a pattern of increasing nucleotide differences for 400
bp between positions +120 to -280 relative to the
initiator codon, followed by 2300 bp of completely different
sequence toward the end of the unique region, and then returning
to a perfect match (for at least 700 bp) with the 801-bp
HHV-8(BC-1) TTR unit sequence (Fig. 4).
The published HHV-8(BC-1) sequence contains 3067 bp of non-TTR
sequence upstream of the initiator codon for ORF75 but is
incomplete at the junction between the unique and TTR domains
because of an unstable (apparently unclonable) G plus A-rich
variable repeat region estimated to be 2-3 kb in size (4). In contrast, our HHV-8(BCBL-R)
sequence contains 2579 bp between the ORF75 initiator codon and
the junction with the first copy of the TTR, which begins at
position 652 within the complete 801-bp TTR sequence given by
Russo et al. (4). There are no G plus
A-repeats at the TTR junction in BCBL-R, but we believe,
nevertheless, that this represents the complete undeleted form
because PCR amplification with primers that span the junction
from the unique region into the RHS TTR gave identical-sized
products from the original BCBL-R tumor DNA sample, the B3-2
phage DNA, and the pDJA62-subcloned plasmid DNA.
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On the basis of our previous identification of three distinct subtypes of HHV-8 in the ORF26 and ORF75 gene regions (18), the published sequence for HHV-8(BC-1) (4) indicates that it represents a C subtype at the RHS end but an A subtype elsewhere, whereas the HHV-8(BCBL-R) genome is an A subtype throughout. DNA from the RHS of our prototype C strain proved to be identical to that in both the BC-1 and HBL6 cell lines in two distinct KS lesions from this patient (ASM72/76) for as far as at least 1200-bp upstream from the ORF75 initiator codon. In contrast, all three other A strains tested (C282, AKS1, and BCBL-1) proved to be nearly identical to BCBL-R between ORF75 and the TTR, although our prototype B strain (431KAP) differed by the presence of both a 10-bp insertion and a 55-bp deletion across the unique sequence/TTR boundary. Therefore, whereas the HHV-8 C strains are dramatically different from the A and B strains over at least 2500 bp (and perhaps up to 5000 bp) at the extreme RHS end of the unique region, B strains proved to be only slightly different from the A strains in this region (Fig. 4
). Interestingly, both the A/B and C strain unique regions upstream and to the right of ORF75 have the potential to encode complex multiply spliced leftwards-oriented mRNAs for hydrophobic membrane proteins that have an equivalent position and orientation to that of EBV LMP-2. Attempts to characterize the very different ORF-K15A/B and ORF-K15C transcripts and gene products are in progress.
Terminal Heterogeneity on the Left-Hand Side
We also carried out plasmid and PCR primer-based sequencing over
a 1500-bp block from genomic nucleotide positions 40-1540,
encompassing the entire ORF-K1 and the C-terminal segment of ORF4
for BCBL-R (pDJA61) and eight other HHV-8 samples from various
sources. Although this segment of the conserved ORF4 coding
region showed only three nucleotide variations over 400 bp among
A, B, and C strains, which is equivalent to the level found for
ORF26 and ORF75 (i.e., approximately 1% differences; 18),
the 840-bp (289 amino acid [aa]) ORF-K1 coding region
proved to display much greater variability (Figs. 4, 5, and 6). Most dramatically, our prototype B strain
HHV-8(431KAP) differed by 14.6% (127 positions) at the DNA
level and 29% (84 amino acids) at the protein level from
the published data for HHV-8(BC-1). Similarly, our prototype C
strain HHV-8(ASM72/76) differed by 5.8% (49 positions) at
the DNA level and 14% (39 amino acids) at the protein
level from HHV-8(BC-1). The B and C strains also differed from
each other at 125 nucleotide positions (and 83 amino acids),
although again the subtype C DNA genomes present in two distinct
lesions from the same patient (ASM72 and ASM76) were identical
throughout. The HBL6 and BC-1 cell lines were derived
independently from the same patient and both are A subtype
sequences throughout (except at the RHS or ORF-K15 region) and as
expected they proved to have identical ORF-K1 DNA sequences.
Among the other A strains tested, BCBL-R exhibited 19-bp
(2.0%) and 13 amino acid (4.5%) changes, BCBL-1
gave 27-bp (2.8%) and 22 aa (8%) changes, and C282
gave 16-bp (1.7%) and 11 aa (4%) changes relative
to HHV-8(BC-1). However, the ORF-K1 regions in BCBL-R, C282,
AKS1, AKS2, and AKS4 differed by only 2-5 bp relative to one
another and all are considered to be members of the A1 substrain
or clade. Even the junctions between the LHS unique sequences and
the TTR differed significantly between BCBL-R(A1), BCBL-1(A3),
and BC1(A2), but no data about the LHS junctions are available as
yet for the prototype B and C strains (Fig. 4). Summary diagrams comparing these ORF-K1 variation
levels with those in the three previously analyzed more conserved
regions in ORF26, ORF75(N), and ORF75(C) (18) are presented in Fig. 5.
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The potential 289 aa membrane protein encoded by the predicted unspliced ORF-K1 gene is a positional and orientation counterpart to LMP-1 of EBV (Fig. 1
), but it lies in
an inverted orientation relative to the highly variable STP (and
TIP) transforming proteins of HVS, and it does not closely
resemble any of these known gamma herpesvirus transformation
related proteins in structure or amino acid content. The fact
that all versions tested remain in frame, despite the presence of
both a 15- and a 6-bp deletion in the prototype C strain, attests
to the presumed functional importance of the ORF-K1 gene product.
Interestingly, the major features of 12 Cys residues and 8
N-glycosylation sites within the N-terminal 175 amino acids of
ORF-K1 are nearly fully conserved and hydrophobic blocks between
positions 1-22 and positions 227 and 251, which are not altered
significantly by the strain variations, probably serve as a
signal peptide and a membrane anchoring transmembrane domain,
respectively (Fig. 6, A). Therefore, the
bulk of the ORF-K1 protein has the appearance of an extracellular
receptor that displays a similar level of variation to that
encountered in the HIV ENV protein. In contrast, the smaller
C-terminal cytoplasmic domain is identical between most A and C
strains, but differs by 30% at the amino acid level in the
B strains. An illustration of the extent of the amino acid
changes over the two most variable extracellular domains of the
ORF-K1 protein between amino acids 52-92 and between 191 and 228
is shown in Fig. 6, B. |
Discussion and Conclusions |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion and ConclusionsReferences |
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Although the recently characterized herpesvirus KSHV or HHV-8 (6) has not yet been demonstrated directly to be transmissible from tumor cells to other target cells or from patient to patient, most of the epidemiologic, serologic, and molecular biology evidence currently available suggests that it is likely to be an essential infectious causative agent of both classical and AIDS-associated KS (29-34). Herpesvirus particles can be induced from cultured HHV-8-positive EBV-negative BCBL cell lines (25) and both the lytic and latent HHV-8-encoded abundant RNAs (T1.1 and T0.7) can be detected in spindle cells and mononuclear cells in KS lesions (8,14,35,36). The genome of the virus is essentially intact and probably circular in the BCBL cell lines, but these points are less clear for the KS tumor versions (9,36,37).
Whether virus-infected cells only provide cytokine support for the other cells in KS lesions or are themselves capable of proliferating is also the subject of considerable debate at present. However, the presence, expression, and functional integrity of the many captured cellular cDNA homologues identified enhance the plausibility that this virus, like EBV and HVS, probably has the potential to directly influence the proliferation state of its host cells whilst in a latent or nonpermissive mode. Expression of the viral-encoded MIP-1-like inflammatory chemokines (even if it occurs only during lytic cycle infection) implies that infected cells may be able to profoundly alter neighboring cells, even to the extent of influencing patterns of HIV infectivity through blocking of the CCR5 co-receptor (11,13,39,40). Furthermore, it has long been known that IL-6 and related cytokines play a major role in the biology of KS spindle cells (41-43), as well as in multiple myeloma (44,45) and MCD (46), and recent evidence by in situ hybridization (Cannon J, Ambinder R: personal communication) and immunohistochemistry (11) that vIL-6 is expressed in vivo in most BCBL tumor cells greatly increases the likelihood that such scenarios may be valid.
Currently, first generation serologic assays for the level of seropositivity to HHV-8 antigens have indicated that the virus may not be widespread outside the AIDS epidemic, except in isolated pockets in areas associated with classical and endemic KS, including some Mediterranean and middle east countries, such as Italy and Greece, and in equatorial Africa, including Uganda and Zaire (24,26,30,31,35,47). Such a pattern would be very atypical for a well-established herpesvirus, whereas one usually expects a ubiquitous distribution of a highly infectious agent with nearly universal asymptomatic primary infection at a very young age. Therefore, many intriguing questions about the origins and mode of transmission of the virus arise. If infection with the virus in humans was actually an ancient and widespread event, then numerous distinct isolates or strains would be expected compared with just a single narrowly diverged genome pattern if it had been recently acquired from an exogenous primate source. Similarly, the patterns of genome heterogeneity observed might be expected to reflect whether the virus in patients with AIDS represents a new horizontally transmitted agent, rather than multiple reactivated endogenous latent infections.
Our evidence clearly shows that there are at least three major
subdivisions of HHV-8 genomes present in human populations (all
three of which are represented in patients with AIDS) and that
(with few exceptions) each patient isolate studied to date can be
distinguished from that of other patients. Importantly, no
differences have yet been found between the genomes present in
multiple lesions from the same patient (10) or in subcloned BCBL cell lines with different passaging
history from the same tumor (i.e., HBL6 and BC1). Our collection
of genomes that were tested came from a wide variety of sources,
but interpretation and extrapolation of the current strain
variability data are greatly complicated by large differences in
the rates of evolutionary divergence in some genes (e.g., ORF-K1)
compared with the others and the existence of two distinct
alternative RHS ends. The possible presence of chimeric or
recombinant genomes containing gene blocks derived from more than
one strain lineage is also implied by mismatches between the
ORF-K1 subtype and the constant region (ORF26/ORF75) subtypes in
some genomes. However, it is not clear as yet whether the low
level of variability in the constant region genes (especially
ORF26) is sufficient to provide a valid measure of subtype
category (Fig. 5). Remarkably, the pattern
and degree of amino acid variability in the HHV-8 ORF-K1 protein
quite closely resemble those seen in both the HIV ENV gene and in
immunoglobulin variable regions, although obviously in this case
the source of the variability and the nature of the selective
pressures involved are at present a complete mystery. Recent
direct PCR screening of HHV-8-positive samples from 22 different
U.S. patients has revealed only five with the C-type of RHS
compared with 17 of the A type. In contrast, eight were of the C
type in the ORF-K1 region on the LHS and 14 were A type, but only
three genomes were found to be C type at both the LHS and RHS
ends. Surprisingly, no B-strain types have been found as yet
amongst either KS or BCBL samples from U.S. patients.
Overall, the results strongly imply that HHV-8 is an ancient human virus with variability and subtype patterns that closely resemble those seen in both EBV and HVS. Whether the A, B, and C strain groupings represent ancient human population bottlenecks, current biologic niche or disease associated selection, or current (pre-AIDS) geographic isolation remains to be determined. However, the fact that five of six KS samples tested from Africa have B strain ORF-K1 regions and that four of our five New York/Baltimore/Washington area AIDS KS samples are closely related A1 strains, whereas the fifth, which was derived from a classic non-AIDS-associated case (EKS1), had a C strain ORF-K1 gene, suggests that we might be able to define clades of the virus similar to the situation in HIV. Obviously, the surprisingly high genetic divergence identified in ORF-K1 among HHV-8 genomes will now provide opportunities to address these questions of origins, geographic preferences, transmissibility, and disease association in considerable detail.
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Acknowledgments |
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Supported by Public Health Service grants CA73585 (G. S. Hayward) and CA06973 (G. S. Hayward and J. Nicholas) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. L. J. Poole is a member of the Biochemistry, Cellular and Molecular Biology training program at The Johns Hopkins University School of Medicine (T32 GM07445). R. T. Sarisky and J. Nicholas were supported in part by a Postdoctoral Fellowship and a Junior Faculty Award, respectively, from the American Cancer Society.
We thank Sarah Heaggans for her assistance in the preparation of the manuscript and diagrams and Skip Virgin and Sam Speck for providing MHV68 sequence data prior to publication.
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