JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1229-12350095-1137/90/061229-07$02.00/0Copyright ©D 1990, American Society for Microbiology

Procaryotic Expression of Phosphorylated Tegument Protein pp65 ofHuman Cytomegalovirus and Application of Recombinant

Peptides for Immunoblot AnalysesB. PLACHTER,' S. KLAGES,'t S. HAGELMANN,' W. BRITT,2 M. P. LANDINI,3 AND G. JAHNl*

Institut fur Klinische und Molekulare Virologie, Universitat Erlangen-Nurnberg, Loschgestrasse 7, D-8520 Erlangen,Federal Republic of Germany'; Department of Pediatrics, University ofAlabama at Birmingham, Birmingham,

Alabama 352942; and Institute of Microbiology, St. Orsola General Hospital, 40138 Bologna, Italy3

Received 13 December 1989/Accepted 21 February 1990

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons,termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immuneresponse during natural infection. A monoclonal antibody directed against this protein was used to screen a

lambda gtll clINA library for recombinant polypeptides. Two DNA fragments from purified lambda clonesand one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. Theresulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed againstpp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodiestested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids.Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen forserodiagnosis but may be very useful in combination with other HCMV proteins.

Human cytomegalovirus (HCMV) is a major cause ofsevere disease in congenitally infected infants (40) and inpatients with impaired immune defenses, such as transplantrecipients or patients suffering from the acquired immuno-deficiency syndrome (9). Early diagnosis of an acute HCMVinfection in these patients is critical with respect to theefficacy of antiviral therapy. Since clinical signs of infectionare often absent in the early stages of infection, quick andreliable laboratory diagnosis is needed.Although virus isolation from patient specimens still is the

most sensitive and specific diagnostic procedure for verify-ing an ongoing acute infection with HCMV, this method isoften time-consuming and laborious. Moreover, virus excre-tion may persist for months to years without being the causeof a specific clinical syndrome. Recently, several methodshave been described for the direct detection of HCMVantigen (1, 41, 42) or HCMV nucleic acid (16, 24, 26, 27, 30,38, 39, 43) in tissues or cells from patients. The specificantibody response against HCMV as a diagnostic marker canbe measured by a variety of commercially available tests.However, these test systems still rely on poorly definedantigens of the virus. Several HCMV proteins have beenstudied in terms of their potential to elicit an antibodyresponse during natural infection (6, 14, 17, 20, 21, 25, 32,33, 37, 44). One phosphorylated tegument protein, pp65 (28),also called lower matrix protein (12), gp64 (5), or ICP27 (11),was reported to be synthesized early in infection (3, 8, 34).This protein is a major constituent of the virus particle andrepresents over 90% of the protein mass of dense bodies,defective particles seen in HCMV-infected cell cultures (13).Several studies have been aimed at the question of whetherthis protein might be a good candidate as a reagent forcytomegalovirus diagnosis (17, 20, 21, 32, 33, 44). However,the results of these experiments cannot be compared directly

* Corresponding author.t Present address: Max-Planck-Institut für Biochemie, D-8033

Martinsried, Federal Republic of Germany.

because the antigen preparations used in the studies were

diverse. A clear statement about the usefulness of pp65 ofHCMV as a reliable antigen for diagnosis, especially for thedetection of the early stages of an acute infection, has thusfar not been possible.The objective of this study was to test defined recombi-

nant proteins carrying epitopes from pp65 with selectedHCMV-positive sera for their reactivity in immunoblots. Toobtain such probes, we screened a lambda gtll expressionlibrary with a monoclonal antibody against pp65; the DNAfragments from the reactive bacteriophages were reclonedinto bacterial high-level expression vectors. The recombi-nant proteins were used to define the binding sites formonoclonal antibodies directed against pp65 and, in a firstset of immunoblot experiments with patient sera, to verifythe antigenic properties of pp65 with respect to the humoralimmune response during natural infection.

MATERIALS AND METHODS

Viruses, cell culture, and virion purification. Laboratorystrain AD169 of HCMV was provided by U. Krech, St.Gallen, Switzerland. Propagation of the virus and purifica-tion of HCMV virions or dense bodies from infected primaryhuman foreskin fibroblast cells were done as describedpreviously (37).Monoclonal antibodies and antisera. For primary screening

of the HCMV cDNA expression library (22) and reactions inimmunoblotting experiments, monoclonal antibody 28-103,directed against the lower matrix protein, pp65, was used(2). Moreover, 12 additional monoclonal antibodies withknown specificities against pp65 were tested in immunoblotsfor reactivity against recombinant proteins. As negativecontrols, monoclonal antibody 28-4, known to be directedagainst the major capsid protein of HCMV (4), and mono-clonal antibody p63-27, known to be reactive with thenuclear immediate-early antigen of HCMV, were used.Polyvalent rabbit antisera were generated by injecting NewZealand White rabbits with preparations of either dense

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bodies or partially purified extracellular particles from cellcultures. In primary injections, 200 ,ug of total protein masswas administered intramuscularly together with incompleteFreund adjuvant. Booster injections were carried out thesame way in about 3-week intervals. Sera were drawn about10 days after each injection. For the immunoblot analyses,several human sera positive for immunoglobulin G (IgG)antibodies and for IgM antibodies against HCMV in enzyme-linked immunosorbent assays (ELISAs) were used.

Immunofluorescence. For indirect immunofluorescence,human foreskin fibroblast cells were grown on glass coverslips and infected with HCMV AD169. After methanolfixation, monoclonal antibodies were layered over the cellsfor 30 min at 37°C. Fluorescein-conjugated rabbit anti-mouseimmunoglobulin (Dakopatts, Hamburg, Federal Republic ofGermany) was added for 30 min (1:40 dilution). After eachincubation step, cells were washed extensively with phos-phate-buffered saline.

Screening of the lambda gtll library and expression clon-ing. Screening of the lambda gtll library with monoclonalantibody 28-103 was done as described previously (17). Theviral inserts of two separate clones were excised with EcoRIand recloned in the bacterial high-level expression vectorpSEM (a kind gift from S. Knapp, Behringwerke, Marburg,Federal Republic of Germany). This vector contains 375amino acids of the bacterial ,-galactosidase gene underhomologous promoter control 5' to a multiple cloning site,giving rise to fusion proteins upon induction with isopropyl-P-D-thiogalactoside. For transformation, Escherichia coliD29A1 (a kind gift from M. Broker, Behringwerke, Marburg,Federal Republic of Germany) was used. The recombinantplasmids were sequenced as described by Sanger et al. (36),and the sequences were compared with the sequence pub-lished for pp65 (35). For the immunoblot analyses, fusionproteins were induced and partially purified as describedpreviously (4).

Protein gel electrophoresis and immunoblotting. Proteinswere separated by 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis by the procedure of Laemmli (19).Transfer of proteins onto nitrocellulose strips and incubationsteps were done as described before (37), with the exceptionthat staining was done with horseradish peroxidase-coupledanti-human IgG, anti-mouse IgG, or anti-rabbit IgG andeither 4-chloro-1-naphthol-H202 or 3,3'-diaminobenzidine.For the immunoblots, murine monoclonal antibodies andmonospecific rabbit antisera were diluted 1:100; humanantisera were diluted 1:50.

In some instances, the rabbit sera were preincubated priorto the immunoblot analyses to reduce background stainingdue to reactions with E. coli proteins. This was done on icefor 1 h in a 5-ml suspension containing a mixture of an E. colilysate, expressing only the ,B-galactosidase portion of thefusion protein (pSEM alone), and 5 ,ug of P-galactosidase(Sigma, Deisenhofen, Federal Republic of Germany). Aftercentrifugation in a Sorvall SS34 rotor (10,000 rpm) for 10min, the supernatant was saved and the preincubation stepwas repeated. AUl other methods were done by standardprocedures (23).

RESULTS

Expression cloning of pp65. The lower matrix protein ofHCMV, also called pp65, can be found in the nuclei ofinfected fibroblasts at early and late times after infection (2).Typical immunofluorescence staining can be seen in Fig. 1.Monoclonal antibody 28-103, directed against pp65, was

FIG. 1. Indirect immunofluorescence of HCMV-infected humanforeskin fibroblast cells 24 h postinfection with monoclonal antibody28-77 directed against pp65 (2). Typical nuclear staining can be seen,corresponding to the localization of pp65 within infected cells.

used to screen a lambda gtll expression library. Twobacteriophage clones were identified in the primary screen-ing and isolated by dilution and replating.DNA from these clones was prepared, and the viral insert

was excised with EcoRI. The fragments were recloned in allthree possible reading frames in the bacterial expressionvector pSEM. The nucleotide sequences of the recombinantclones were determined in part and compared with thepublished sequence of the pp65 gene (35). One clone, termedpSEML68-2, was found to contain viral sequences fromnucleotides 1539 to 1749 of the published sequence (Fig. 2),thus expressing 70 amino acids of pp65 (Fig. 3, lane 4). Thesecond clone, p68-1, extended from nucleotides 888 to 1758.To clone the nonoverlapping region of p68-1 with pSEML68-2, we recloned an EcoRI-HindIII fragment of 588 base pairsfrom p68-1, extending from nucleotides 888 to 1476 (Fig. 2),in pSEM. The resulting clone, termed pHE68-1, expressed astable fusion protein of about 69 kilodaltons (kDa) (Fig. 3,lane 3). Additionally, a small AvaII-NcoI fragment of 78nucleotides was cloned out of the coding region forpSEML68-2 and expressed in pSEM as clone pSK5. Thisclone was found to contain sequences from nucleotides 1540and 1618 and produced a fusion protein of about 50 kDa (Fig.3, lane 5).

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26 aa

M1618 1540

70 aa

1749 1539

pSK 5

pSEML68-2

196 aa

1476 888pHE68- 1

I .. ORF PP65

o ac) -_rA

r.m0

Fl cb a d F- i rI lo N J M F D L -U||P S R T E | K Q|X|V H HindIII

K SQ E IMIPM I |A D B R JOL C TN EcoRIhf 9g de

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 map units

0 25 50 75 100 125 150 175 200 225 250 kb

FIG. 2. Schematic representation of the prototype arrangement of the HCMV AD169 genome and the locations of the gene fragments frompp65 used for expression cloning. Restriction sites are given for the endonucleases EcoRI and HindIII. HindIII fragments b and c are shownon an expanded scale. The stippled box marks the location of the open reading frame (ORF) coding for pp65. The solid bars indicate thelocations of the DNA fragments coding for the viral parts of the fusion proteins. The numerals above these bars indicate the numbers of aminoacids (aa) from pp65 present in each expression clone; the numerals below the bars represent the nucleotides of the viral sequence (numberingaccording to Ruger et al. [35]). kb, Kilobases.

Western blot (immunoblot) analyses of the recombinantproteins with monoclonal antibodies and polyvalent rabbitantisera. To verify that the recombinant fusion proteins fromclones pHE68-1, pSEML68-2, and pSK5 were recognized byantibodies known to react with viral pp65, we carried outWestern blot analyses. Recombinant fusion proteins were

separated on denaturing sodium dodecyl sulfate-polyacryla-mide gels. After transfer to nitrocellulose strips, the recom-binant proteins were incubated in a first set of experimentswith monoclonal antibody 28-103. This monoclonal antibodyhad been used for the initial lambda screening. The antibodyreacted with the fusion proteins of clones pSEML68-2 andpSK5 but not with the fusion protein of clone pHE68-1 (Fig.4A, lanes 1 to 3). It also reacted with the band of about 65kDa in the virion preparation (Fig. 4A, lane 5). These resultsclearly indicate that (i) the monoclonal antibody detects an

epitope in the recombinant pp65 proteins and that (ii) thisepitope for monoclonal antibody 28-103 is located within theoverlapping region of the fusion proteins from clonespSEML68-2 and pSK5 (Fig. 2).A panel of 13 different murine monoclonal antibodies

directed against pp65 was also used for immunoblot analyseswith the recombinant proteins. All reacted with fusionproteins from pSK5 and pSEML68-2, as well as the pp65

band in virion preparations (data not shown). The results ofthese experiments suggest that one epitope is located withinthe 26 amino acids encoded by the viral insert of pSK5,which appears dominant in mice for the elicitation of anti-bodies against pp65.

In a second set of experiments, the recombinant proteinswere reacted with rabbit antisera raised against gel-purifiedpp65 and against the dense-body fraction (17), as well as withrabbit antisera raised against purified extracellular particlesof HCMV. All these sera preferentially reacted with thefusion proteins from clones pSEML68-2 and pSK5, as wellas with the viral protein of 65 kDa. One experiment with a

polyvalent rabbit antiserum is shown in Fig. 4B. In addition,a rabbit antiserum was raised by injecting a partially purifiedfusion protein from clone pSK5; this antiserum also reactedwith the fusion proteins from pSK5 and pSEML68-2, as wellas with the viral band of 65 kDa (data not shown).Western blot analyses of the recombinant proteins with

human sera. To resolve the discrepancies about the reactiv-ity of pp65 (17, 20, 21, 44), we tested a number of human sera

with our recombinant fusion proteins in immunoblots. Eachstrip contained the three recombinant proteins from pp65and a fusion protein from expression clone pXP1. This clonerepresents a portion of another tegument protein (ppl50)

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FIG. 3. Polyacrylamide gel analysis of lysates from E. coliD29A1 expressing the fusion proteins from pp65. Lanes: 1, molec-ular weight markers; (molecular weights at left in thousands); 2,lysate containing 375 amino acids of ,B-galactosidase expressedwithout a viral insert; 3, lysate containing a fusion protein fromexpression clone pHE68-1; 4, lysate containing a fusion protein fromexpression clone pSEML68-2; 5, lysate containing a fusion proteinfrom expression clone pSK5. Protein extracts were loaded on a 10%polyacrylamide gel and stained with Coomassie brilliant blue.

which was shown to be consistently reactive with HCMV-positive human sera (37).

In a first set of experiments, we addressed the question ofwhether the recombinant proteins from pp65 would reactwith human sera known to have a high antibody titer againstHCMV. For this purpose, two panels of sera were used. Onepanel consisted of five serum specimens from our diagnosticlaboratory. These sera all contained IgM antibodies againstHCMV, as measured by an ELISA. In this panel, all serareacted with the ppl50 recombinant protein. Only three offive serum specimens reacted with one or more of therecombinant proteins from pp65. One representative blot isshown in Fig. SA. A strong reaction was seen with the pXP1fusion protein (lane 5); good reactivity was also seen with thepSEML68-2 fusion protein, and weak reactions were seenwith the fusion proteins from pSK5 and pHE68-1.The second panel consisted of 18 serum specimens also

known to have IgM antibodies. Nine of these serum speci-mens were from patients (either healthy persons or trans-plant recipients) with acute primary HCMV infections, andnine were from renal transplant recipients with acute sec-ondary HCMV infections. Only three of the nine serumspecimens from the primary infections and two of the nineserum specimens from the secondary infections reactedabove the background with pp65 recombinant proteins. Oneexample is shown in Fig. 5B.These results indicate that in a number of the sera from

patients with acute HCMV infections, antibodies against ourpp65 recombinants were not detectable in the immunoblotassay.

----- 69 kDa65 kDa

50 kDa6-5 kD<

FIG. 4. (A) Immunoblot analysis of the recombinant fusion pro-teins with monoclonal antibody 28-103 directed against pp65. Lanes:1, E. coli lysate containing a fusion protein from clone pSK5; 2, E.coli lysate containing a fusion protein from clone pSEML68-2; 3, E.coli lysate containing a fusion protein from clone pHE68-1; 4, E. colilysate containing only the P-galactosidase portion of pSEM (nega-tive control); 5, purified virions from cell culture supernatants. (B)Immunoblot analysis of the recombinant fusion proteins with apolyvalent rabbit antiserum raised against extracellular particles ofHCMV. Lanes: 1, E. coli lysate containing a fusion protein fromclone pSK5; 2, E. coli lysate containing a fusion protein from clonepSEML68-2; 3, E. coli lysate containing a fusion protein from clonepHE68-1; 4, E. coli lysate containing a fusion protein from clonepXP1; 5, E. coli lysate containing only the P-galactosidase portion ofpSEM (negative control); 6, purified virions from cell culturesupernatants; 7, purified extracellular particles from infected-cellcultures. The numbers on the right side of each panel give theapparent molecular masses of the fusion proteins of the pp65 virionband.

To exclude the possibility that antibodies against pp65 aremade early in infection and decrease in titer rapidly thereaf-ter, thus being undetectable, we used a third panel of humansera kindly provided by Jaap Middeldorp. This panel con-sisted of 16 sequential serum specimens from five transplantrecipients with either acute primary or acute secondaryinfections with HCMV (four primary infections and onesecondary infection, as measured by the presence of IgGantibodies by an ELISA or seroconversion from negative toIgG positive, respectively). The sera were tested as de-scribed above. The results obtained agreed with those fromthe first set of experiments. A reaction against pp65 recom-binant proteins was seen in 5 of 13 ELISA-positive serumspecimens (data not shown). All of these serum specimenswere from patients with acute primary infections. In none ofthese primary infections were antibodies against the pp65recombinant proteins detectable before the appearance ofantibodies against the pplS0 recombinant protein.

DISCUSSION

A number ofHCMV proteins which react with patient serafrom different stages of clinical infections in immunoblotsand radioimmune precipitations have been described (15, 17,

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FIG. 5. (A) Immunoblot analysis of the recombinant fusion pro-

teins with a human serum containing IgG and IgM antibodies againstHCMV. Lanes: 1, E. coli lysate containing a fusion protein and

expression clone pSK5; 2, E. coli lysate containing a fusion proteinfrom expression clone pSEML68-2; 3, E. coli lysate containing a

fusion protein from expression clone pHE68-1; 4, E. coli lysate

containing only the P-galactosidase portion of pSEM (negative

control); 5, E. coli lysate containing a fusion protein from expressionclone pXP1. (B) Immunoblot analysis of the recombinant fusion

proteins with a serum specimen from a renal transplant recipientwith an acute primary infection with HCMV. Lanes: 1, E. coli lysate

containing a fusion protein from expression clone pSK5; 2, E. colilysate containing a fusion protein from expression clone pSEML68-

2; 3, E. coli lysate containing a fusion protein from expression clone

pHE68-1; 4, E. coli lysate containing only the P-galactosidaseportion of pSEM (negative control). The fusion protein of

pSEML68-2 showed partial degradation in this assay, represented

by the appearance of smaller reactive bands. With this serum

specimen, a strong reaction with two of the pp65 recombinants was

seen. The numbers beside the panels give the apparent molecular

masses of the fusion proteins.

20, 21, 29, 32, 33, 44). The results of these reports are

somewhat controversial in terms of the antigens most reli-

ably detected in certain clinical stages of infection. This may

be due in part to the heterogeneity of the antigen prepara-

tions used. For example, some authors used proteins from

infected cells as sources of antigens, and some used prepa-

rations of extracellular particles. These preparations differ in

that (i) not ail HCMV antigens found in infected celîs are also

present in extracellular particles and in that (ii) the relative

amounts of certain proteins prepared from infected ceils are

distinct from those found in extracellular particles. For

instance, it is well known that two of the major tegument

phosphoproteins, pp7l and ppl50, are underrepresented in

antigen preparations from infected cells as compared with

preparations of extracellular virions (34). Additionally, even

the results of those studies in which extracellular particlesfrom celi culture supernatants were used as antigens cannot

be compared directly, because different purification proce-

dures lead to different amounts of dense-body particles.These particles consisted of .:90% pp65 (13). Their presence

in antigen preparations may result in a distorted picture of

the relative intensity of the humoral immune response

against this protein.A second factor which makes it difficult to compare the

results of such studies is that different detection systems forthe antibody reactions were used. Some authors carried outradioimmune precipitations, thus being able to detect anti-gen-antibody interactions depending on native proteins (15,28, 29, 32, 33, 44). Others used the immunoblot system, withwhich linear epitopes can be assessed efficiently (14, 17, 20,21, 37, 44). These differences and the fact that estimates ofthe molecular weights of certain proteins differed because ofthe gel system used caused considerable confusion about theHCMV proteins dominant in eliciting a humoral response inthe host.One way to resolve this dilemma is to clearly define the

antigens on the molecular level. This way is, however,hampered by the fact that only a few genes coding for thestructural components of HCMV have thus far been charac-terized. One ofthese is the gene coding for pp65. The codingregion for this protein on the genome of HCMV had beendetermined before (28, 31), and the nucleotide sequence hadbeen established (35). It was found to be distinct from that ofanother phosphorylated protein of about 67 kDa also de-scribed in preparations of extracellular virions (7).The objective of this study was to clone and express parts

of pp65 in bacterial high-level expression vectors to obtaindefined antigens representing immunodominant epitopes ofthis protein. Three recombinants representing about 50% ofthe open reading frame for pp65 were obtained. With this setof recombinant proteins, we were able to study the immunereactivity against specific epitopes of pp65 independent ofany potentially interfering reactivity against epitopes ofother HCMV proteins similar in size. Furthermore, by beingable to use defined amounts of antigens we achieved betterreproducibility and comparability of the results.

In one series of immunoblots, we showed that 14 murinemonoclonal antibodies with known specificities against pp65reacted with the fusion protein of clone pSK5. The corre-sponding region of pp65 between amino acids 401 and 426can be considered an immunodominant one in mice.

In a second series of immunoblots, polyvalent rabbitantisera were tested for reactivity with the three recombi-nant proteins. Sera drawn as early as 10 days after the thirdinjection with viral antigen were positive in the immuno-blots. The reaction was directed against ail three fusionproteins, as well as against the band of about 65 kDa presentin the virion preparation. These results prove that pp65 is anantigenic protein and that antibodies directed againstepitopes outside of pSK5 also elicit an antibody response.The same result was seen when a polyclonal rabbit serumraised against a dense-body preparation was used for West-ern blotting. However, the strong and early antibody re-sponse seen in the rabbits when an antigen preparation witha high proportion of pp65 was used does not reflect thesituation during natural infection in humans, as infectiousvirions from recent clinical isolates do not necessarily con-tain pp65 as a major constituent (18). This difference mightexplain why high-titered human sera were shown not to reactconsistently with the pp65 band from virion preparations inWestern blots (17).

Essentially the same results were obtained in this workwith human sera drawn during acute HCMV infections whenrecombinant polypeptides were used as antigens. Only alimited number of the sera used reacted with the three pp65recombinant proteins, as compared with the reactivityagainst one recombinant protein from ppl50. This resultindicates that antibodies against pp65 are less reliable indetecting acute HCMV infections. This result also agreeswith the findings ofGibson and Irmiere, who stated that pp65

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-- 72 kDa

- 65 kDhô

- 50 kDa

FIG. 6. Immunoblot analysis of nonspecific reactivities of unre-lated monoclonal antibodies or HCMV ELISA-negative humansera. (A) Immune reaction of monoclonal antibody p63-27, directedagainst the immediate-early antigen of HCMV. Lanes: 1, E. colilysate containing a fusion protein from clone pSK5; 2, E. coli lysatecontaining a fusion protein from clone pSEML68-2; 3, E. coli lysatecontaining a fusion protein from clone pHE68-1; 4, E. coli lysatecontaining a fusion protein from clone pXP1; 5, E. coli lysatecontaining only the P-galactosidase portion of pSEM (negativecontrol); 6, purified virions from cell culture supernatants. (B)Immune reaction of a human serum specimen negative in an IgGELISA for antibodies to HCMV. Lanes: 1, E. coli lysate containinga fusion protein from clone pSK5; 2, E. coli lysate containing afusion protein from clone pSEML68-2; 3, E. coli lysate containing afusion protein from clone pHE68-1; 4, E. coli lysate containing afusion protein from clone pXP1; 5, E. coli lysate containing only theP-galactosidase portion of pSEM (negative control); 6, infected-cellproteins from permissive human foreskin fibroblasts harvested at 6days postinfection; 7, purified virions from cell culture supernatants.The numbers on the right side of each panel give the apparentmolecular masses of the fusion proteins, the pp65 band seen invirion preparations and in infected-cell preparations, and the 72-kDaband corresponding to the immediate-early antigen of HCMV. Asexpected, the last band was seen in infected cells but not in virions.

is less immunogenic in humans than are other HCMVproteins (13). Additionally, one has to consider some non-specific binding of antibodies to pp65, as has been reportedby Britt and Auger (2). We can confirm these findings, as wesometimes saw a nonspecific reaction in Western blots withrecombinant proteins and HCMV-negative human sera andalso with some totally unrelated monoclonal antibodies (Fig.6).The tegument protein pp65 might be one major target of

the cell-mediated immune response during natural infectionwith HCMV (10). It also causes a humoral immune responsein animals as well as in humans. The results of this study,however, indicate that pp65 alone is not a reliable antigen touse as a diagnostic tool but might be very helpful in combi-nation with other antigens for the detection of acute stages ofHCMV infections. A study with a larger panel of HCMV-positive human sera will define the optimal antigen combi-nation to be used for HCMV diagnosis.

ACKNOWLEDGMENTS

We thank Michael Mach for providing the HCMV gtll library.The technical assistance of Gabi Buttner and the photographic workof W. Rossler are gratefully appreciated.

This work was supported by the Wilhelm-Sander-Stiftung and theBundesministerium für Forschung und Technologie (Projekt-Nummer 01KI8806).

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1 23456A

-69 kDa-65 kDa

- 50 kDa

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