Department of Clinical and Experimental Medicine, Division of Microbiology, Laboratory of Virology, University of Bologna, St Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy1
Author for correspondence: Maria Paola Landini. Fax +39 051 341632. e-mail viroland{at}med.unibo.it
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Recently, a novel structural protein of CMV was identified as the product of the gene UL25 (EMBL accession no. X17403) and was localized within the viral tegument (Baldick & Shenk, 1996 ; Battista et al., 1999
; Zini et al., 1999
). Because of its post-translational modification (phosphorylation), its localization in the tegument and its molecular mass (approximately 85 kDa), we investigated whether the product of UL25 corresponded to the antibody-reactive structural protein of approximately 82 kDa.
The Towne strain of CMV was used for all experiments. The virus was propagated in human embryo fibroblasts by using standard methods and purified by sorbitol cushion and sorbitol gradient as described previously in detail (Gleaves et al., 1984 ). Purified viral particles were lysed in PAGE loading buffer by boiling in the presence of
-mercaptoethanol and viral p82 (vp82) was separated and purified by preparative PAGE with a Prep Cell (Bio-Rad) (Lazzarotto et al., 1998
).
The construction of UL25 prokaryotic expression plasmids has been described previously in detail (Battista et al., 1999 ). In the construct, UL25 was inserted in the EcoRI site of the vector pCKS between the 5 and 3 sequences encoding the bacterial protein CMP-2-keto-3-deoxyoctulosonic acid synthetase (CKS) under the control of the lacZ promoter. Recombinant pUL25 (rpUL25) was purified by Q-Sepharose chromatography (Pharmacia Biotech) after lysis by a combination of detergent washes followed by solubilization in 8 M urea (Robinson et al., 1993
).
Both vp82 and rpUL25 were used in blots to determine the binding of specific antibodies by using a procedure described recently (Lazzarotto et al., 1998 ). Briefly, a sheet of nitrocellulose was inserted into a miniblotter device (Biometra). Individual suspensions of vp82, rpUL25 and other viral and recombinant proteins in 50 mM TrisHCl, 5 mM EDTA (pH 9), were deposited onto the nitrocellulose. The Escherichia coli CKS protein was added as a negative control to monitor the presence of serum IgM and IgG to the bacterial portion of the fusion protein. Human µ chain and
chain were added as positive controls to monitor the reaction of the conjugate to human IgM and IgG. Miniblotters were gently agitated overnight at room temperature on a rocking platform. The filters were washed briefly in Tris-buffered saline (TBS) and then saturated by incubation with a blocking solution (3% fish gelatin, 1% BSA, 5% powdered skimmed milk, 0·05% Tween 20 in TBS) at room temperature for 1 h. The filters were then cut in 3 mm-wide strips. The final amount of protein per strip was 50 ng vp82, 60 ng rpUL25, 25 ng CKS and 25 ng µ and
chain.
A total of 466 sera from 320 subjects were used in this work. More precisely, 200 sera (group A of Table 1) were from 200 blood donors (obtained courtesy of the Blood Transfusion Centre of St Orsola General Hospital, Bologna, Italy), 50 (group B of Table 1
) were from 50 IgM-positive immunocompetent subjects (mainly youths and pregnant women), 106 sera (group C of Table 1
) were from 50 pregnant women undergoing active CMV infections and finally 110 samples (group D of Table 1
) were from 20 solid-organ transplant recipients who underwent CMV infection during the first 5 months after transplantation (10 patients had a primary infection while 10 underwent virus reactivation).
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Diagnosis of active virus infection was carried out by the antigenaemia test and/or PCR on polymorphonuclear leukocytes for immunocompromised patients (Revello et al., 1989 ; van der Bij et al., 1988
), while, in the case of pregnant women, the detection of one or more of the following parameters was considered indicative: virus isolation from urine, saliva or blood, seroconversion for anti-CMV antibodies and presence of low-avidity antibodies (Lazzarotto et al., 1999
).
Serum samples were diluted 1:50 in TBS with 10% foetal calf serum (FCS) and 100 µg/ml CKS. Incubation was performed at room temperature for 1 h. After three washes with TBSTween 20 (0·05% Tween 20 in TBS), a goat anti-human µ and chainalkaline phosphatase conjugate (kindly provided by Abbott Laboratories) diluted 1:1500 in TBS with 5% FCS was added and the mixture was incubated at room temperature for 1 h. Washing and development were performed as described previously (Lazzarotto et al., 1998
).
As shown in Table 1, most of the subjects showing either IgG or IgM to vp82 also reacted positively with pUL25. In particular, among 320 subjects (466 sera) tested we had only nine discordant results (1·9%). In eight cases, reactivity was observed for vp82 and not for rpUL25. In six of these eight cases, reactivity to the recombinant protein was obtained at a lower serum dilution, indicating that a minor portion of the reactivity could be addressed to eukaryotic-specific post-translational modifications. We also had one case of reactivity to rpUL25 not correlated with reactivity to vp82. This serum had antibodies to the carrier protein that could not be removed satisfactorily. Furthermore, CMV-positive sera with no antibody to vp82 did not react with pUL25.
In view of the good concordance between serum reactivity to vp82 and rpUL25, we affinity-purified antibodies specific for rpUL25 and verified their ability to bind vp82 and, vice versa, affinity-purified antibodies specific for vp82 and verified their binding to rpUL25. In the first experiment, nitrocellulose with blotted proteins from induced bacterial extracts was stained with Ponceau S and the strip containing the fusion protein CKSpUL25 was excised. The strip was washed several times to remove the red stain, saturated with the blocking solution and incubated overnight with two pooled CMV-positive sera. After extensive washing, antibodies were eluted with 0·2 M glycineHCl, pH 2·5, neutralized with 1 M potassium phosphate, pH 9·0, and immediately used as probes on nitrocellulose blots of purified virus and purified vp82. In the second experiment, a strip of nitrocellulose containing purified vp82 was used for affinity purification of IgG from two pooled CMV-positive sera. Eluted antibodies reactive with vp82 were then used as probes on blots containing electrophoretically separated proteins from purified virus particles and purified rpUL25. The results are shown in Fig. 1. Antibodies affinity-purified on rpUL25 can bind vp82 and those affinity-purified on vp82 bind rpUL25.
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Acknowledgments |
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References |
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Received 18 July 2000;
accepted 18 October 2000.
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