Department of Medical Microbiology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
Correspondence
Cornelis Vink
kvi{at}lmib.azm.nl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleotide and amino acid sequences discussed in this paper have been deposited in the GenBank database under accession number AF232689.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our laboratory, we are studying the interaction between rat CMV (RCMV) and its host as a model for HCMV infection and disease (Bruggeman et al., 1982). RCMV contains a linear, double-stranded DNA genome of 230·1 kb. The complete DNA sequence of the RCMV genome has been determined recently and was found to contain at least 167 open reading frames (ORFs) (Vink et al., 2000
). Most of these ORFs have counterparts in the genomes of both HCMV and murine CMV (MCMV) (Chee et al., 1990
; Rawlinson et al., 1996
; Vink et al., 2000
). However, an exception is RCMV ORF r127, which is unique among the CMVs. This ORF has the capacity to encode a 337 amino acid protein (pr127) which shows similarity to the non-structural proteins (NS or Rep proteins) that are encoded by the rep genes of parvoviruses (Vink et al., 2000
). The predicted amino acid sequence of the r127-encoded protein is most closely related to the sequences of the Rep1/2 proteins of three avian parvoviruses, namely barbary duck parvovirus (BDPV), muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) (Vink et al., 2000
; Zadori et al., 1995
). Although the RCMV rep gene homologue is unique among the CMVs, it is not unique among the betaherpesviruses: the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B) also show similarity to the parvoviral rep genes (Dominguez et al., 1999
; Gompels et al., 1995
; Isegawa et al., 1999
; Thomson et al., 1991
). The U94 ORF was first discovered in HHV-6A and was found to encode a 490 amino acid protein (RepH6) that is most closely related to the Rep proteins of the adeno-associated viruses (AAVs), including Rep68/78 of AAV-2 (Srivastava et al., 1983
; Thomson et al., 1991
). Interestingly, a counterpart of U94 has not been identified in the genome of HHV-7, which is closely related to HHV-6A and -6B (Nicholas, 1996
). Remarkably, although RCMV r127 and HHV-6A and -6B U94 have a conserved genomic position as well as orientation (Dominguez et al., 1999
; Gompels et al., 1995
; Isegawa et al., 1999
; Vink et al., 2000
), rep gene homologues have so far not been found in the genomes of other herpesviruses.
The best characterized rep gene product is Rep68/78 of AAV-2 (reviewed by Berns, 1996). Rep68/78 is involved in many aspects of the virus replication cycle of AAV-2. It is required for viral DNA replication, modulation of viral and cellular gene expression and site-specific integration of the viral genome into human chromosome 19. Rep68/78 possesses several activities that correspond to its role in the virus replication cycle, including sequence-specific DNA-binding activity, site- and strand-specific endonuclease activity and ATP-dependent helicase activity (Berns, 1996
).
The role of RepH6 in the virus replication cycles of HHV-6A and -6B is less well documented. However, RepH6 seems to have a conserved function with its AAV-2 counterpart, since HHV-6A U94 is able to complement the replication of a rep-deficient AAV-2 genome (Thomson et al., 1994). In addition, U94 is transcribed in latently infected peripheral blood mononuclear cells (PBMCs) from HHV-6A-infected individuals (Rotola et al., 1998
), suggesting a role for RepH6 in the regulation of latency. This possibility is supported by the fact that virus replication and expression of viral genes are restricted in HHV-6A-infected lymphocytes expressing HHV-6B U94 in vitro (Rotola et al., 1998
). Nevertheless, not much is known about the role of RepH6 in the pathogenesis of viral infection. Moreover, it is not possible to study the role of RepH6 in animal models. We therefore set out to study the biological function of pr127, the RepH6 homologue of RCMV. Here, we show that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity. Furthermore, we demonstrate that the r127-encoded protein is dispensable for virus replication in vitro and in vivo.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Poly(A)+ RNA isolation from RCMV- and mock-infected REFs.
Poly(A)+ RNA was isolated from RCMV-infected REFs at immediate early (IE), early (E) and late (L) times of infection. During the 1 h infection period, the REFs were exposed to RCMV at an m.o.i. of 1·0. IE mRNA was extracted from REFs that had been treated with 100 µg cycloheximide ml1 from 1 h prior to infection until they were harvested at 16 h post-infection (p.i.). E mRNA was obtained from REFs that had been treated with 200 µg phosphonoacetic acid ml1 from 3 h p.i. until harvesting at 16 h p.i., and L mRNA was isolated from REFs that were harvested at 72 h p.i. Mock mRNA was extracted from REFs that had not been infected with RCMV. Poly(A)+ RNA was purified with the QuickPrep Micro mRNA purification kit (Amersham Biosciences) and dissolved in RNase-free H2O. mRNA was quantified by determination of the absorbance at 260/280 nm.
Northern blot analysis.
Northern blot analysis was performed on 5 µg portions of mRNA from RCMV- and mock-infected REFs (see above). Samples were separated by electrophoresis through an agarose/formaldehyde gel, blotted onto a Hybond-N nylon membrane (Amersham Biosciences) and hybridized to a DNA probe representing the full-length r127 ORF. This probe was generated after amplification of ORF r127 by PCR using primers 5'-r127 (5'-ACGTGGATCCATGAAGACTAGAACCGG-3') and 3'-r127 (5'-ACGTAAGCTTAACATCCTAGTCAC-3'). These primers introduced unique BamHI and HindIII restriction sites into the amplified fragment. Construct pRXO, which contains the RCMV XbaI O fragment (Meijer et al., 1986), was used as target DNA. The amplified fragment of 1·0 kb was digested with BamHI and HindIII and cloned into the corresponding restriction sites of cloning vector pUC119, generating plasmid p302. The sequence of ORF r127 was confirmed by DNA sequencing using the Thermo Sequenase cycle sequencing kit (Amersham Biosciences) with Cy5-labelled M13 universal primers and an ALFexpress automated DNA sequencer (Amersham Biosciences). Construct p302 was then digested with BamHI and HindIII and the resulting fragment containing ORF r127 was labelled with [
-32P]dATP (ICN) using the Random Primed DNA labelling kit (Roche Applied Science). The radioactive signal was visualized by autoradiography.
5'- and 3'-RACE.
Rapid amplification of 5' and 3' cDNA ends (5'- and 3'-RACE) was performed on L mRNA (see above) using the SMART RACE cDNA amplification kit (BD Biosciences) and the Marathon cDNA amplification kit (BD Biosciences). Gene-specific primers r127P4 (5'-GGACTCCCGGGTTCCCAAGTACCTC-3'; position 178973 to 178997 of the RCMV genome; Vink et al., 2000) and r127P3 (5'-AGATGCGAGTCCCCGGTCGATAAAC-3'; position 179164 to 179140) were used for the 5'- and 3'-RACE, respectively. Amplified fragments were cloned into vector pGEM-T Easy (Promega) and their sequence was determined by DNA sequencing, as described above.
Total RNA isolation from tissues of RCMV-infected rats.
Three-week-old, male Wistar rats (Central Animal Facilities, University of Maastricht) were infected by intraperitoneal injection of 1x105 p.f.u. RCMV and sacrificed at either 1 week or 4 months p.i. Salivary gland, spleen, kidney, liver and lung tissues were excised and total RNA was isolated as described previously (Gauthier et al., 1997). RNA was dissolved in RNase-free H2O and quantified by determination of the absorbance at 260/280 nm.
Generation of in vitro-transcribed RNA.
Construct p302 (see above) was digested with BamHI and HindIII and the resulting 1·0 kb fragment containing ORF r127 was cloned into BamHI- and HindIII-digested vector pGEM-3Z (Promega), generating plasmid p271. In this construct, ORF r127 is located downstream of the T7 RNA polymerase promoter. A 1·2 kb fragment of construct p271 containing the T7 promoter and ORF r127 was amplified by PCR using M13 universal primers. A sample (0·5 µg) of the amplified fragment was transcribed in the presence of T7 RNA polymerase (Amersham Biosciences) and subsequently treated with DNase I (Amersham Biosciences). RNA was purified by extraction with phenol/chloroform, recovered by ethanol precipitation, dissolved in RNase-free H2O and quantified by determination of the absorbance at 260/280 nm.
RT-PCR.
RT-PCR was performed on 0·5 µg samples of total RNA from tissues of RCMV-infected rats (see above). Each sample was denatured for 5 min at 65 °C in the presence of 0·4 µg tRNA µl1 (Roche Applied Science). Samples were then chilled on ice and added to 50 µl pre-chilled reaction mixture containing 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 3 mM MgCl2, 2 mM DTT, 0·2 mM each dNTP, 10 U RNAguard (Amersham Biosciences), 0·25 U HotStarTaq DNA polymerase (Qiagen), 5 U SuperScript II reverse transcriptase (Invitrogen) and 0·2 µM each of primer P3 (5'-CGTATCGTCTGATCCGAACC-3'; position 178908 to 178889 of the RCMV genome) and P4 (5'-GGGATCTCTCCACCAGATGA-3'; position 178661 to 178680). Each sample was reverse transcribed for 20 min at 50 °C. Subsequently, the HotStarTaq DNA polymerase was activated for 15 min at 95 °C. The reverse-transcribed target was then amplified by 40 cycles of denaturation for 30 s at 94 °C, primer annealing for 30 s at 59 °C and primer extension for 30 s at 72 °C. A 1 µl aliquot of the RT-PCR product was subjected to another 30 cycles of PCR with nested primers P1 (5'-CCATAACCTCAACCCTCGTG-3'; position 178889 to 178870 of the RCMV genome) and P2 (5'-CTACACAGGCAGCCAGGTCT-3'; position 178690 to 178709). Each sample was also processed in the absence of reverse transcriptase to monitor any residual DNA contamination. Furthermore, two additional reactions were run in parallel for each sample. In one of these reactions, the sample was spiked with 1020 copies of in vitro-transcribed RNA (see above) to address whether possible contaminants interfered with the amplification. In the other reaction, the sample was subjected to RT-PCR with rat -actin gene-specific primers RT-ACT-B (5'-GGTGGGTATGGGTCAGAAGG-3') and RT-ACT-F (5'-TGCCGATAGTGATGACCTGA-3') to confirm the integrity of the sample.
Generation of MBPpr127 and 6Hpr127 expression constructs.
Construct p302 (see above) was digested with BamHI and HindIII and the resulting 1·0 kb fragment containing ORF r127 was cloned into the corresponding restriction sites of expression vectors pMAL-c (New England Biolabs) and pRSET A (Invitrogen), generating plasmids p248 and p232. These constructs encode proteins containing the complete r127-derived amino acid sequence fused to the C terminus of either maltose-binding protein (MBPpr127; construct p248) or a tag consisting of six consecutive histidine residues (6Hpr127; construct p232).
Expression and purification of MBPpr127 and MBP-gal-
.
Construct p248 (see above) and expression vector pMAL-c were introduced into Escherichia coli BL21(DE3)pLysS and the resulting strains were grown overnight at 37 °C in LB medium containing 50 µg ticarcillin ml1 and 20 µg chloramphenicol ml1. The cultures were diluted 1 : 100 in 300 ml LB medium with ticarcillin and chloramphenicol and grown at 37 °C to an OD600 of 0·6. Protein expression was then induced by the addition of IPTG to a final concentration of 0·3 mM. After 3 h of protein expression at 37 °C, the bacteria were harvested by centrifugation and resuspended in 10 ml of buffer A (10 mM sodium phosphate buffer, pH 7·2, 1 mM EDTA, 1 mM -mercaptoethanol) supplemented with 1 M NaCl. The suspensions were sonicated and cleared by centrifugation. To the supernatants, 10 ml of buffer A was added. The materials were then loaded onto 2 ml amylose columns (New England Biolabs). The columns were washed with 7·5 ml buffer B (buffer A supplemented with 0·5 M NaCl) containing 0·25 % Tween 20 and subsequently with 7·5 ml buffer B. Proteins were eluted with 7·5 ml buffer B containing 10 mM maltose. Fractions of 0·5 ml eluted protein were collected and analysed by SDS-PAGE. Peak fractions were pooled, dialysed into buffer B containing 10 % glycerol and stored at 80 °C.
Generation of rabbit polyclonal antibodies directed against MBPpr127.
A rabbit was immunized by intramuscular injection of 1 mg purified MBPpr127 (see above) in Specol adjuvant (Animal Sciences Group, Wageningen University and Research Centre). An intramuscular booster injection, containing 1 mg purified MBPpr127, was given at week 9. Blood was obtained before and 13 weeks after immunization, and sera were prepared as described previously (Harlow & Lane, 1988). The reactivity of these sera against the RCMV pr127 protein was determined by Western blot analysis.
Western blot analysis.
Constructs p248 and p232 (see above) and expression vector pMAL-c were introduced into E. coli BL21(DE3)pLysS and protein expression was induced essentially as described above. Proteins from crude bacterial extracts were separated by SDS-PAGE and transferred to a PROTRAN BA 83 nitrocellulose membrane (Schleicher & Schuell). The blots were then incubated with a 1 : 1000 dilution of either rabbit anti-MBPpr127 antiserum or rabbit pre-immune serum (see above) and subsequently with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulins (DakoCytomation). The blots were stained with diaminobenzidine.
Immunocytochemical analysis of RCMV- and mock-infected Rat2 cells.
During a 1 h infection period, Rat2 cells were either mock-infected or infected with RCMV at an m.o.i. of 0·1. The cells were fixed and permeabilized at 8, 12, 24 and 72 h p.i. The cells were then incubated with a 1 : 100 dilution of either rabbit anti-MBPpr127 antiserum or rabbit pre-immune serum (see above) and subsequently with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulins (DakoCytomation). The fluorescent label was visualized with an Axiovert 100 fluorescence microscope (Zeiss). Staining of cells with monoclonal antibody RCMV8 was carried out as described previously (Bruning et al., 1987; Kaptein et al., 2001
).
Immunohistochemical analysis of tissues of RCMV- and mock-infected rats.
Ten-week-old, male Brown Norway rats (Central Animal Facilities, University of Maastricht) were immunocompromised by 5 Gy of total-body Röntgen irradiation 1 day prior to infection, as described previously (Stals et al., 1990). Rats were either mock-infected or infected by intraperitoneal injection of 3x105 p.f.u. RCMV. The animals were sacrificed at 3 weeks p.i. and 4 µm paraffin tissue sections were prepared from salivary gland, spleen and liver. Serial tissue sections were mounted on glass slides and deparaffinized. Sections were then incubated with a 1 : 100 dilution of either rabbit anti-MBPpr127 antiserum or rabbit pre-immune serum (see above) and subsequently with biotin-conjugated swine anti-rabbit immunoglobulins (DakoCytomation) and streptavidinbiotinylated alkaline phosphatase (AP) complex (DakoCytomation). Sections were stained with Fast Red (Speel et al., 1992
). Staining of sections with monoclonal antibody RCMV8 was performed as described previously (Bruning et al., 1987
; Kaptein et al., 2001
).
DNA-binding assay.
Aliquots of 150 µg of either purified MBPpr127 or MBP-gal-
(see above) in 1 ml binding buffer (10 mM Tris/HCl, pH 7·4, 25 mM KCl, 0·5 mM EDTA, 0·05 % Tween 20, 100 mM NaCl) were added to 1 ml single- and double-stranded DNA-cellulose columns (Amersham Biosciences). The columns were washed with 5 ml binding buffer and proteins were eluted with 0·5 ml fractions of binding buffer supplemented with 0, 100, 200, 400, 600, 800, 1000 and 1500 mM NaCl. Fractions of eluted protein were collected and analysed by SDS-PAGE.
Generation of an RCMVr127 recombination plasmid.
Vector pRc/CMV (Invitrogen) was digested with XhoI and the resulting 2·1 kb fragment containing a neomycin resistance gene (neo) was cloned into SalI-digested pBluescript SK (+) vector (Stratagene), generating plasmid p474. Construct p474 was then digested with ClaI and XhoI and the 2·1 kb neo fragment was used to replace the 0·5 kb ClaIXhoI fragment within ORF r127 of construct pRXO. The resulting RCMVr127 recombination plasmid was designated p475.
Generation of an RCMV r127 deletion mutant.
Approximately 2x107 Rat2 cells were trypsinized and harvested by centrifugation. The cells were washed and resuspended in 500 µl of serum-free culture medium. To the cell suspension, 20 µg of construct p475 (see above) was added. The suspension was transferred to a 0·4 cm electroporation cuvette (Bio-Rad) and pulsed at 0·25 kV and 500 µF in a Gene Pulser electroporator (Bio-Rad). The transfected cells were subsequently seeded in culture flasks. At 16 h after transfection, the cells were infected with low-passage RCMV at an m.o.i. of 1·0. The culture medium was supplemented with 50 µg G418 ml1 at 24 h p.i. Recombinant virus was plaque-purified and cultured on REFs as described previously (Beisser et al., 1998, 1999
, 2000
; Kaptein et al., 2003
). The integrity and plaque purity of the RCMV r127 deletion mutant (RCMV
r127) were determined by Southern blot analysis.
Southern blot analysis.
DNA was isolated from wild-type (wt) RCMV- and RCMVr127-infected REFs and digested with XbaI and XhoI. The digested samples were separated by electrophoresis through an agarose gel and blotted onto a Hybond-N+ nylon membrane (Amersham Biosciences). Constructs pRXO and p474 (see above) were used as RXO- and neo-specific probes, respectively. These constructs were labelled with the DIG DNA labelling kit. The DIG Easy Hyb solution was used for hybridization, and the DIG wash and block buffer set and the DIG luminescent detection kit (all from Roche Applied Science) were used for detection.
Replication of wt RCMV and RCMVr127 in vitro.
During a 1 h infection period, REFs were infected with either wt RCMV or RCMVr127 at an m.o.i. of 0·01. At days 1, 3, 5 and 7 p.i., culture medium samples were taken and subjected to plaque assays. The data were statistically analysed by applying Student's t-test. P values of <0·05 were considered to indicate statistical significance.
Replication of wt RCMV and RCMVr127 in vivo.
Two groups of ten 7-week-old, male, specific-pathogen-free (SPF) Lewis/M rats (Central Animal Facilities, University of Maastricht) were immunocompromised by 5 Gy of total-body Röntgen irradiation at 1 day prior to infection, as described previously (Stals et al., 1990). Rats were infected by intraperitoneal injection of 1x106 p.f.u. of either wt RCMV or RCMV
r127. At days 4 and 28 p.i., 5 rats from each group were sacrificed. Salivary gland, spleen, kidney, liver, pancreas and thymus tissues were collected and subjected to plaque assays. The data were statistically analysed by applying Student's t-test. P values of <0·05 were considered to indicate statistical significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The 1·1 kb r127 transcript that was identified by the RACE experiments (Fig. 2c) is likely to correspond to the 1·3 kb mRNA that was detected by Northern blot analysis (Fig. 2a
). We therefore conclude that the RCMV r127 gene is transcribed during the E and L phases of virus replication in vitro as an unspliced transcript of approximately 1·1 kb, comprising the full-length r127 ORF.
The r127 gene is transcribed during RCMV infection in vivo
To examine whether the r127 gene is transcribed during the acute phase of RCMV infection in vivo, an r127-specific, nested RT-PCR assay was performed on total RNA purified from salivary gland, spleen, kidney, liver and lung of RCMV-infected rats at either 1 week or 4 months p.i. The assay, which has a lower detection limit of approximately 10 copies of RNA (data not shown), was designed to amplify a 200 bp fragment. At 1 week p.i., r127-specific transcripts were detected in all five organs tested (Fig. 2d, lanes 812). As expected, transcripts of r127 were not detected when RNA was omitted from the reaction mixture (Fig. 2d
, lane 13). Furthermore, amplified fragments were derived from RNA rather than from contaminating DNA, since they were not generated when samples were processed in the absence of reverse transcriptase (Fig. 2d
, lanes 27). Spiking each sample with in vitro-synthesized RNA and subjecting each sample to an RT-PCR assay with a primer set specific for the rat
-actin gene confirmed the efficiency of all enzymic reactions and the integrity of all RNA samples (data not shown). At 4 months p.i., r127-specific transcripts were only detected in the salivary gland and not in spleen, kidney, liver or lung (data not shown). This pattern of r127 transcription parallels the temporal production of infectious virus in organs of RCMV-infected rats (Bruggeman et al., 1985
). Taken together, the RCMV r127 gene is widely transcribed during productive infection, both in vitro and in vivo.
Generation of rabbit polyclonal antibodies directed against the RCMV pr127 protein
In order to study expression of the RCMV r127-encoded protein, we set out to generate rabbit anti-pr127 polyclonal antibodies. To this end, an MBPpr127 fusion protein was expressed in E. coli and purified by affinity chromatography (Fig. 3a, upper gel). The purified protein (Fig. 3a
, upper gel, lanes 715) had a calculated molecular mass of 80 kDa and was approximately 90 % pure. Minor proteins in the MBPpr127 preparation with molecular masses lower than 80 kDa probably represented degradation products of the full-length fusion protein. Peak fractions of purified MBPpr127 (Fig. 3a
, upper gel, lanes 815) were pooled, dialysed and used to immunize a rabbit. The reactivity of the resulting rabbit anti-MBPpr127 antiserum against pr127 was tested by Western blot analysis (Fig. 3b
). This antiserum reacted with MBPpr127 (Fig. 3b
, lane 2) as well as with 6Hpr127 (lane 3). As might be expected, reactivity was also seen with a protein containing the bacterial
-galactosidase-
protein fused to MBP (MBP
-gal-
; Fig. 3b
, lane 4), but not with a protein containing part of the RCMV IE1 protein fused to a combined 6Hthioredoxin tag (6HTRXIE1; lane 5) (Beuken et al., 1999
). Rabbit pre-immune serum did not react with any of these proteins (Fig. 3b
, lanes 710). These data clearly indicate that the rabbit anti-MBPpr127 antiserum contains antibodies directed against both MBP and pr127. Since eukaryotic cells do not express MBP, this antiserum is a useful tool to study expression of the pr127 protein in RCMV-infected cells.
|
In conclusion, the pr127 protein is a nuclear protein that is expressed as early as 12 h p.i. in RCMV-infected cells in vitro. The kinetics of pr127 expression are in accordance with the early-late kinetics of r127 transcription (see above).
The pr127 protein is expressed within the nuclei of RCMV-infected cells in vivo
To examine in vivo expression of the pr127 protein during the acute phase of RCMV infection, immunohistochemical analysis using the rabbit anti-MBPpr127 antiserum (see above) was performed on salivary gland, spleen and liver of RCMV-infected rats at 3 weeks p.i. As shown in Fig. 3(d), pr127 is expressed within the nuclei of RCMV-infected cells in all three organs tested (panels AC). As expected, specific staining was not observed either in corresponding tissue sections from mock-infected rats (data not shown) or after incubation with rabbit pre-immune serum (Fig. 3d
, panels DF). As shown previously (Bruning et al., 1987
; Kaptein et al., 2001
), staining with anti-pR44 monoclonal antibody RCMV8 also resulted in nuclear staining of RCMV-infected cells in these organs (data not shown). Taken together, the pr127 protein is widely expressed within the nuclei of RCMV-infected cells during productive infection, both in vitro and in vivo.
The RCMV pr127 protein has single- and double-stranded DNA-binding activity
There are several indications that the RepH6 proteins of HHV-6A and -6B might be involved in the regulation of viral and/or cellular gene expression. One of these is the ability of HHV-6B RepH6 to bind single-stranded DNA (Dhepakson et al., 2002). To determine whether the RCMV pr127 protein also possesses DNA-binding activity, we tested its capacity to bind to DNA-cellulose. First, fusion proteins MBPpr127 (80 kDa) and MBP
-gal-
(51 kDa) were purified from E. coli, as described above (Fig. 3a
). The purified proteins were then tested for their affinity for single- and double-stranded DNA-cellulose columns. As shown in Fig. 4
, MBPpr127 was found to bind single- (a, lane 4) as well as double-stranded DNA-cellulose (c, lane 4), although its affinity for single-stranded DNA-cellulose seemed somewhat more pronounced. Since MBPpr127 does not bind to cellulose alone (data not shown), we conclude that the affinity of this protein for DNA-cellulose is the result of DNA binding by MBPpr127. The DNA-binding activity of this protein appears to be strong, given that the protein was only partially eluted from the columns with increasing concentrations of NaCl (Fig. 4a and c
, lanes 513). Furthermore, the DNA-binding activity of MBPpr127 was pr127-specific, since the columns did not retain MBP
-gal-
(Fig. 4b and d
, lane 4). These results indicate that the RCMV pr127 protein has single- as well as double-stranded DNA-binding activity.
|
|
The r127 gene is dispensable for RCMV replication in vivo
To study the role of the pr127 protein in the pathogenesis of RCMV infection, we compared the in vivo replication characteristics of wt RCMV and RCMVr127 during productive infection by monitoring their dissemination in infected rats. The amount of infectious virus produced in salivary gland, spleen, kidney, liver, pancreas and thymus of wt RCMV- and RCMV
r127-infected rats was determined at 4 and 28 days p.i. Table 2
shows that there was no significant difference between wt RCMV and RCMV
r127 in tissue distribution at both 4 and 28 days p.i. High virus titres were predominantly detected in the salivary glands of infected rats at 28 days p.i. As shown in Fig. 5(d)
, these titres did not differ significantly between wt RCMV and RCMV
r127. The unlikely possibility that, within the RCMV
r127-infected rats, the recombinant virus was overgrown by contaminating wt RCMV, e.g. because of insufficient plaque purifications, was excluded by Southern blot analysis on DNA purified from virus that was derived from salivary gland homogenates of RCMV
r127-infected rats at 28 days p.i. (data not shown).
|
Taken together, the RCMV r127 gene is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, not much is known about the role of RepH6 in the pathogenesis of HHV-6A and -6B infection. Nevertheless, the U94 genes of HHV-6A and -6B are highly conserved (Rapp et al., 2000), which indicates that RepH6 may play an important role in the replication cycles of these viruses. Interestingly, RepH6 seems to share at least some function with its parvoviral counterparts, since HHV-6A U94 can complement the replication of a rep-deficient AAV-2 genome (Thomson et al., 1994
). In addition, U94 is transcribed in latently infected PBMCs from HHV-6A-infected individuals, whereas both virus replication and expression of viral genes are restricted in HHV-6A-infected lymphocytes expressing HHV-6B U94 in vitro (Rotola et al., 1998
). These observations point to a potential role for RepH6 in the regulation of latency. RepH6 may be involved in either establishment or maintenance of latency through a mechanism involving the regulation of gene expression. This notion is based on the observations that this protein can bind to the human TATA-binding protein, that it binds single-stranded DNA and that it is able to regulate expression from several promoters (Araujo et al., 1995
, 1997
; Dhepakson et al., 2002
; Mori et al., 2000
; Thomson et al., 1994
).
At this point, we do not know whether pr127 has a similar role in the pathogenesis of RCMV infection. Although we found pr127 to share characteristics with RepH6, such as DNA-binding activity, we were unable to pinpoint a specific function for pr127 in RCMV replication. Furthermore, it is highly unlikely that pr127 has the same ability as RepH6 to complement parvoviral Rep proteins, since pr127 is considerably shorter than RepH6. More specifically, in comparison with the parvoviral Rep proteins, both pr127 and RepH6 are truncated at their C termini, while pr127 is also truncated at its N terminus (Vink et al., 2000). Nevertheless, although we found RCMV
r127 to have replication characteristics indistinguishable from those of wt RCMV during the acute phase of infection in vivo, we cannot rule out the possibility that these viruses present with different features in the initiation and maintenance of latency. Due to the unavailability of an appropriate, reproducible experimental model in which to study RCMV latency and reactivation, we have not yet been able to investigate the role of pr127 in these biological processes. Although we detected transcripts of r127 at 4 months p.i. in the salivary glands of RCMV-infected rats, this does not represent latent gene expression, as infectious virus is still produced in the salivary glands at 4 months p.i. (Bruggeman et al., 1985
). It is clear, however, that the development of a model for RCMV latency and reactivation will have a high priority in future studies.
Regardless of its potential role in RCMV latency, it is to be expected that pr127 does have a function during the acute phase of infection. This notion is inferred from the finding that this protein is expressed during productive infection, both in vitro and in vivo. The function of pr127 may have been overseen, because either (i) the function is subtle, (ii) it is only apparent in specific host strains or (iii) we did not use a correct model system. Our future studies will be aimed at the elucidation of the physiological role of the nuclear localization and DNA-binding activity of pr127 and, more specifically, at the identification of viral and/or cellular proteins that bind to pr127. These studies will be likely to shed more light on the role of pr127 in RCMV replication.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Araujo, J. C., Doniger, J., Stoppler, H., Sadaie, M. R. & Rosenthal, L. J. (1997). Cell lines containing and expressing the human herpesvirus 6A ts gene are protected from both H-ras and BPV-1 transformation. Oncogene 14, 937943.[CrossRef][Medline]
Beisser, P. S., Vink, C., van Dam, J. G., Grauls, G., Vanherle, S. J. V. & Bruggeman, C. A. (1998). The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J Virol 72, 23522363.
Beisser, P. S., Grauls, G., Bruggeman, C. A. & Vink, C. (1999). Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain. J Virol 73, 72187230.
Beisser, P. S., Kloover, J. S., Grauls, G. E. L. M., Blok, M. J., Bruggeman, C. A. & Vink, C. (2000). The r144 major histocompatibility complex class I-like gene of rat cytomegalovirus is dispensable for both acute and long-term infection in the immunocompromised host. J Virol 74, 10451050.
Berns, K. I. (1996). Parvoviridae: the viruses and their replication. In Fields Virology, 3rd edn, vol. 2, pp. 21732197. Edited by B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman & S. E. Straus. Philadelphia, PA: Lippincott-Raven.
Beuken, E., Grauls, G., Bruggeman, C. A. & Vink, C. (1999). The rat cytomegalovirus R32 gene encodes a virion-associated protein that elicits a strong humoral immune response in infected rats. J Gen Virol 80, 27192728.
Bruggeman, C. A., Meijer, H., Dormans, P. H. J., Debie, W. M. H., Grauls, G. E. L. M. & van Boven, C. P. A. (1982). Isolation of a cytomegalovirus-like agent from wild rats. Arch Virol 73, 231241.[Medline]
Bruggeman, C. A., Meijer, H., Bosman, F. & van Boven, C. P. A. (1985). Biology of rat cytomegalovirus infection. Intervirology 24, 19.[Medline]
Bruning, J. H., Debie, W. H. M., Dormans, P. H. J., Meijer, H. & Bruggeman, C. A. (1987). The development and characterization of monoclonal antibodies against rat cytomegalovirus induced antigens. Arch Virol 94, 5570.[Medline]
Chee, M. S., Bankier, A. T., Beck, S. & 12 other authors (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125169.[Medline]
Dhepakson, P., Mori, Y., Jiang, Y. B., Huang, H. L., Akkapaiboon, P., Okuno, T. & Yamanishi, K. (2002). Human herpesvirus-6 rep/U94 gene product has single-stranded DNA-binding activity. J Gen Virol 83, 847854.
Dominguez, G., Dambaugh, T. R., Stamey, F. R., Dewhurst, S., Inoue, N. & Pellett, P. E. (1999). Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A. J Virol 73, 80408052.
Gauthier, E. R., Madison, S. D. & Michel, R. N. (1997). Rapid RNA isolation without the use of commercial kits: application to small tissue samples. Pflugers Arch 433, 664668.[CrossRef][Medline]
Gompels, U. A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B. J., Martin, M. E. D., Efstathiou, S., Craxton, M. & Macaulay, H. A. (1995). The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology 209, 2951.[CrossRef][Medline]
Harlow, E. & Lane, D. (1988). In Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Isegawa, Y., Mukai, T., Nakano, K. & 10 other authors (1999). Comparison of the complete DNA sequences of human herpesvirus 6 variants A and B. J Virol 73, 80538063.
Kaptein, S. J. F., Beuken, E., Grauls, G. E. L. M., Bruggeman, C. A. & Vink, C. (2001). Rat cytomegalovirus open reading frame R44 is an early-late gene that encodes a nuclear protein. Arch Virol 146, 22112218.[CrossRef][Medline]
Kaptein, S. J. F., Beisser, P. S., Gruijthuijsen, Y. K., Savelkouls, K. G. M., van Cleef, K. W. R., Beuken, E., Grauls, G. E. L. M., Bruggeman, C. A. & Vink, C. (2003). The rat cytomegalovirus R78 G protein-coupled receptor gene is required for production of infectious virus in the spleen. J Gen Virol 84, 25172530.
Lukashov, V. V. & Goudsmit, J. (2001). Evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses. J Virol 75, 27292740.
Meijer, H., Dreesen, J. C. F. M. & van Boven, C. P. A. (1986). Molecular cloning and restriction endonuclease mapping of the rat cytomegalovirus genome. J Gen Virol 67, 13271342.[Abstract]
Mori, Y., Dhepakson, P., Shimamoto, T., Ueda, K., Gomi, Y., Tani, H., Matsuura, Y. & Yamanishi, K. (2000). Expression of human herpesvirus 6B rep within infected cells and binding of its gene product to the TATA-binding protein in vitro and in vivo. J Virol 74, 60966104.
Nicholas, J. (1996). Determination and analysis of the complete nucleotide sequence of human herpesvirus 7. J Virol 70, 59755989.[Abstract]
Rapp, J. C., Krug, L. T., Inoue, N., Dambaugh, T. R. & Pellett, P. E. (2000). U94, the human herpesvirus 6 homolog of the parvovirus nonstructural gene, is highly conserved among isolates and is expressed at low mRNA levels as a spliced transcript. Virology 268, 504516.[CrossRef][Medline]
Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 88338849.[Abstract]
Rotola, A., Ravaioli, T., Gonelli, A., Dewhurst, S., Cassai, E. & Di Luca, D. (1998). U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture. Proc Natl Acad Sci U S A 95, 1391113916.
Speel, E. J. M., Schutte, B., Wiegant, J., Ramaekers, F. C. S. & Hopman, A. H. N. (1992). A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-Fast Red reaction. J Histochem Cytochem 40, 12991308.
Srivastava, A., Lusby, E. W. & Berns, K. I. (1983). Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol 45, 555564.[Medline]
Stals, F. S., Bosman, F., van Boven, C. P. A. & Bruggeman, C. A. (1990). An animal model for therapeutic intervention studies of CMV infection in the immunocompromised host. Arch Virol 114, 91107.[Medline]
Tatusova, T. A. & Madden, T. L. (1999). BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174, 247250.[CrossRef][Medline]
Thomson, B. J., Efstathiou, S. & Honess, R. W. (1991). Acquisition of the human adeno-associated virus type-2 rep gene by human herpesvirus type-6. Nature 351, 7880.[CrossRef][Medline]
Thomson, B. J., Weindler, F. W., Gray, D., Schwaab, V. & Heilbronn, R. (1994). Human herpesvirus 6 (HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2) and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression. Virology 204, 304311.[CrossRef][Medline]
Vink, C., Beuken, E. & Bruggeman, C. A. (2000). Complete DNA sequence of the rat cytomegalovirus genome. J Virol 74, 76567665.
Zadori, Z., Stefancsik, R., Rauch, T. & Kisary, J. (1995). Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2. Virology 212, 562573.[CrossRef][Medline]
Received 1 December 2003;
accepted 25 February 2004.