Cells, virus, and human samples.
HCMV strains were propagated in human foreskin fibroblasts (HFF) as previously described and maintained as frozen stocks (41
). Human foreskin fibroblast cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. A low-passage clinical isolate was used (49) (stock titer 108
PFU/ml). The extraction and purification of HCMV DNA have been described previously (41
). The bone marrow samples were archived samples obtained from the Cleveland Clinic under an approved Institutional Review Board protocol and were sent to us as coded samples. The identity of the donor remains confidential. Blood from the donors used in preparation of the cDNA library was obtained under an approved University of Nevada at Reno Institutional Review Board protocol. The handling of human samples complied with federal guidelines and institutional policies.
Fifteen milliliters of the blood was mixed with 15 ml HistoPaque-1077 cushion and centrifuged at 400 × g for 30 min at room temperature. The supernatant was then removed to reduce the number of contaminating platelets and the remainder was washed twice with 50 ml of Hanks' medium. The resulting white peripheral blood mononuclear cell pellet was resuspended by gentle mixing in Trizol reagent.
Nucleic acid extraction.
Total RNA was isolated from fresh or frozen tissue by using guanidinium isothiocyanate-acid phenol (TRIZOL LS reagent, GIBCO BRL, Rockville, MD) according to the manufacturer's instructions and established methods (8
). Cells were lysed in a 3.5-cm culture dish by adding 0.3 to 0.4 ml of TRIZOL LS reagent and passing the cell lysate through a pipette several times. After incubating the homogenized samples for 5 min at room temperature to obtain complete dissociation of nucleoprotein complexes, 0.2 ml of chloroform was added. Tubes were shaken vigorously by hand for 15 s, incubated at room temperature for 15 min, and centrifuged at 12,000 × g
for 15 min at 4°C.
Following centrifugation, the mixture separated into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase containing RNA. After transferring the aqueous phase to a clean tube, RNA was precipitated by adding 0.5 ml isopropyl alcohol per 0.75 ml of TRIZOL LS reagent used for the initial homogenization. Samples were then incubated at room temperature for 10 min and centrifuged at 12,000 × g for 10 min at 5°C. After removing the supernatant, the RNA pellet was washed once by adding 1 ml of 75% ethanol per 0.75 ml of TRIZOL LS reagent used for the initial homogenization. Samples were mixed on a vortex and centrifuged at 7,500 × g for 5 min at 5°C. The RNA pellet was briefly dried and then reconstituted in RNase-free water.
Poly(A)+ RNA purification.
Poly(A)+ RNA was purified using oligo(dT)-cellulose columns (Molecular Research Center, Inc.) according to the manufacturer's instructions. Total RNA was precipitated with isopropanol and dissolved in binding buffer (0.5 M LiCl, 50 mM sodium citrate, 0.1% sodium dodecyl sulfate) to a concentration of 4 mg RNA/ml and incubated at 70°C for 5 min. The RNA solution was applied to an oligo(dT)-cellulose column (Molecular Research Center, Inc.). The eluate was reintroduced in the column. The column was washed twice with 1 ml of binding buffer (1 mM sodium citrate, 0.1% sodium dodecyl sulfate) and eluted with 0.9 ml of elution buffer (1% sodium dodecyl sulfate, 75% ethanol, 1 M Tris, 5 M LiCl). The RNA concentration was measured spectrophotometrically. RNA was supplemented with LiCl and Polyacryl Carrier (Molecular Research Center, Inc.). The RNA-carrier was precipitated by centrifugation and dissolved in diethyl pyrocarbonate-treated water.
Creation of cDNA library in lambda phage.
Poly(A)+ RNA isolated from monocytes of a healthy donor with a high antibody titer against HCMV was used to construct a cDNA library and directionally cloned into lambda-ZAP (Lambda Zap Synthesis Kit, Stratagene) following the manufacturer's recommendations.
Double-stranded cDNA was synthesized from 5 μg of poly(A)+ RNA with 20 U of Moloney murine leukemia virus reverse transcriptase. Single-stranded DNA containing oligo(dT) and XhoI restriction endonuclease recognition sequences (ZAP cDNA Synthesis Kit; Stratagene) was used as a primer. The sequence of this primer is (GA)10ACTAGTCTCGAG(T)18. The GAGA sequence was added to protect the XhoI restriction site (bold). 5-methyl dCTP was used in the first strand synthesis. The end product of this reaction is a double-stranded 5-methyl cDNA-RNA molecule.
The sample was treated with RNase H to remove of the template RNA, and amplified with the same primer used for the first strand synthesis, single-stranded DNA containing oligo(dT) and XhoI restriction endonuclease recognition sequences. To measure the amount and size distribution of cDNA, an aliquot was amplified in the presence of [α-32P]dATP (Amersham). The end products of this reaction are double-stranded DNA molecules where only one strand is methylated. The cDNA termini were blunt using blunting deoxynucleoside triphosphate mix and cloned Pfu DNA polymerase (ZAP cDNA Synthesis Kit; Stratagene).
The hemimethylated, blunt-ended double-stranded cDNA was ligated to EcoRI linkers and digested with XhoI. Adapted cDNAs were separated from linker fragments by chromatography in an alkaline agarose gel. Column fractions 2 to 13 were collected, pooled, and extracted with phenol-chloroform and ethanol precipitation. After the size distribution was determined by agarose gel electrophoresis and autoradiography, the adapted cDNA was ligated to EcoRI- and XhoI-digested lambda-ZAP arms at 4°C for 48 hours. The ligated phage was packaged in vitro, and the resultant phage titer was determined by limiting dilution plaque forming assay Escherichia coli XL1-Blue MRF′ on NZY agar plates containing 5 mg of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) per ml and 3 μM of isopropylthiogalactopyranoside (IPTG). The stock library was prepared by plate lysate propagation of the phage in E. coli XL1-Blue MRF' on NZY agar plates.
Phage DNA isolation.
The DNA from lambda phage was isolated as previously described (33
). Briefly, DNase and RNase were added to a library aliquot to a final concentration of 100 μg/ml and incubated at 37°C for 30 min. Twenty microliters of 2 M ZnCl2
was added per ml of lysate and incubated for 5 min at 37°C. The sample was pelleted by centrifugation and resuspended in 500 μl of TES (0.1 M Tris-HCl [pH 8], 0.1 M EDTA, 0.3% sodium dodecyl sulfate) and incubated at 65°C for 15 min. Then, 60 μl of 3 M potassium acetate was added, and incubated on ice for 20 min. The sample was pelleted again by centrifugation and the DNA present in the supernatant was ethanol precipitated.
Synthetic oligonucleotide primers (Invitrogen, Inc.) for immediate-early 1 (IE1) HCMV genes (19
), human β actin, and lambda ZAP (Stratagene, Inc.) were selected (Table ). Previously described primers from the literature are also described in Table (18
). Primers for several HCMV genes, within the UL80-UL82 region were designed (Table ). The relative orientation of primers in the UL81-UL82 region is shown in Fig. . The accuracy of every HCMV primer set was confirmed using HCMV DNA as a template.
List of primers for area UL80-82a
Graphic representation of the HCMV genome, relative positions of genes analyzed in this study, and primers and probes used.
PCR amplification and cloning of HCMV-specific transcript cDNAs.
RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen). The program for the reverse transcription (RT)-PCR had an initial step of 1 h at 41°C instead of the initial denaturation. The PCR conditions were initial denaturation at 94°C, 30 cycles of 94°C for 30 seconds, 55 to 58°C (depending of the primers used) for 30 seconds, and 72°C for 1 min, followed by 10 cycles of 94°C for 30 seconds, 55 to 58°C for 30 seconds, and 72°C for 2 min. After the cycling process the products were exposed to final elongation of 72°C for 5 min. In case of the nested PCRs a 2-μl aliquot from the first round of amplification PCR was used to perform the second round of amplification.
Amplified products that did not have enough DNA for direct sequencing were cloned into plasmid vector by TA cloning (Invitrogen, Inc.). The unmodified PCR fragment was ligated to the pCR 2.1-TOPO vector for 5 min at room temperature and transformed in TOP10F' cells (Invitrogen, Inc.). Colonies were picked and grown in 2× YT medium overnight. An aliquot of the liquid culture (500 μl) was heat inactivated at 96°C for 10 min and spun down, and the supernatant was used for PCR using vector primers. Sequencing was done at the Nevada Genomic Center of University of Nevada Reno, using the ABI PRISM 3730 DNA analyzer. Purified PCR products were sequenced using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).
Southern blot analysis.
Southern blot analysis was performed with the following standard procedures. PCR products were separated by electrophoresis on a 1% agarose gel and ethidium bromide. The gel was treated with 0.2 N HCl (5 min), washed three times in diH2O (3 min each time) and treated with 0.4 N NaOH (5 min). The DNA was transferred overnight onto a nylon membrane by capillary blotting in 0.4N NaOH. The probe consisted of a small fragment of DNA corresponding to PCR fragment amplified with primers UL81F2 and UL81R3, starting with HCMV DNA as the template (Fig. ). The PCR product was visualized in an agarose gel stained with ethidium bromide, cleaned and sequenced. The purified DNA fragment corresponding to fragment between primers UL81F2-UL81R3 was labeled with random primer using [α-32P]dCTP (Amersham Pharmacia Biotech Inc.), and used as a probe. The nylon membrane was prehybridized for 1 h at 65°C and hybridized overnight with the described 32P-labeled probe at 65°C. The membrane was washed twice in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate (SDS) at 65°C for 15 min, and twice in 0.1× SSC with 0.1% SDS at 65°C for 30 min. Bands were detected by X-ray film at −80°C after different exposure times according to the experiment design.
Rapid amplification of 3′ cDNA ends.
Total RNA, poly(A)+ RNA and RNA extracted from HCMV-infected cells were used as a template. The RNAs were reverse transcribed using random primers, or specific UL81 primers and Superscript II reverse transcriptase. For completing the 3′ rapid amplification of cDNA ends (RACE), the cDNA was used as a template. Primers from UL81 region were used in combination with adaptor primer (AP, Table ) to complete the first-round PCR. For the second-round PCR, the reaction medium (2 μl) from the first-round PCR was used as a template. Again, primers from the UL81 region were used but this time in combination with the universal adaptor primer (UAP, Table ).
Protein cloning and purification.
An open reading frame (ORF) corresponding to UL82as protein was amplified by PCR using primers TOPO-UL82as F and UL82as R (Table ). To enable directional cloning, the forward primer was designed to contain CACC at the 5′ end, which pairs with the overhang sequence GTGG in the vector. The reverse primer was initiated with the codon preceding LUNA, the putative stop codon (TGA), so that the cloned PCR product produced an in-frame fusion with the C-terminal His tag. PCR constructs were verified by sequencing. The LUNA ORF PCR was cloned into pET102/D TOPO (Invitrogen). Recombinant LUNA was tagged with V5 and His epitopes. Recombinant protein was expressed in Escherichia coli BL21 start cells (Invitrogen) after induction with 1 mM isopropyl-β-d-thiogalactoside in LB for 24 h at 37°C. Cells were harvested by centrifugation. Recombinant LUNA protein was purified using HiTrap Chelating Ni2+ affinity column (Amersham Biosciences) according to manufacturer's protocol.
Bacterial cell pellets were resuspended in resuspension buffer (20 mM Tris-HCl, pH 8.0), using 4 ml of this buffer per 100 ml of initial culture used. Proteases inhibitor cocktail was added to this solution. Three cycles of freeze/thaw were conducted with dry ice and methanol mix and a 37°C water bath. Cells were further lysed using an 18-gauge syringe in between each cycle. Cells were centrifuged at high speed for 10 min at 4°C, and pellets were resuspended cold isolation buffer (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0). A freeze and thaw procedure was repeated and followed by a high-speed centrifugation (10,000 × g) step for 10 min at 4°C. The pellet material was washed once in isolation buffer lacking urea, and resuspended in binding buffer (6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0), using 5 ml of buffer for every 100 ml of initial culture. The samples were incubated at room temperature for 60 min with gentle rocking, followed by a high-speed centrifugation for 15 min at 4°C. Supernatant was filtrated using 0.45-μm filters in order to remove the remaining particles, before applying to the column.
We washed 5 ml HiTrap Chelating columns with 5 ml of distilled water. Columns were loaded with 0.5 ml of 0.1 M NiSO4, and excess salt was washed with 5 ml of distilled water. Approximately 5 to 10 ml of binding buffer (6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0) was used to equilibrate the column before sample loading. After sample loading, the columns were washed with 10 ml of binding buffer, followed by 10 ml of wash buffer (6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0). Proteins were refolded in the column using a linear 6 to 0 M urea gradient. The gradient started with the wash buffer described above and finished with refolding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0). A gradient volume of 30 ml and a flow rate of 0.1-1 ml/min were used. Columns were washed with 5 ml of refolding buffer.
Refolded recombinant LUNA was eluted using a 0 to 1 mM 2-mercaptoethanol linear gradient starting with refolding buffer and ending with elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, 1 mM 2-mercaptoethanol, pH 8.0). A gradient volume of 20 ml and a flow rate of 0.1-1 ml/min were used. Aliquots were collected and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) gels. This procedure purified LUNA-His6 to homogeneity based on Coomassie-stained SDS-PAGE (data not shown). For Coomassie staining, the gels were placed in a solution containing 0.25% Coomassie brilliant blue (Sigma) in 45% methanol, 10% acetic acid for 2 h to overnight and washed in the same solution with no dye. Western blot analysis also detected LUNA and the His tags.
Polyclonal antibodies against purified recombinant LUNA were raised in rabbits. Two New Zealand female rabbits were used to obtain the polyclonal antibodies in sequential immunization every month, for 4 months. Before every immunization, blood samples were taken from the marginal vein of the rabbit ear, centrifuged and the sera were frozen. The immune sera were used in Western blot and immunoprecipitation experiments. Preimmune sera were used as negative controls.
Serum purification (reabsorption).
The following is an adaptation of protocol described by Streit and Stern (42
Preparation of insoluble cell powder.
Human fibroblast cells were cultured in several T175 flasks to 100% confluence. Cells were washed with Hanks' balanced salt solution, trypsinized, and collected by centrifugation. Cell pellets were dissolved in four volumes of ice-cold acetone, and incubated on ice for 30 min. Cell solutions were spun down at 10,000 × g for 10 min, and cell pellets were washed once with 4 volumes of ice-cold acetone. Cell pellets were spread out on Whatman filter paper, and grinded to a fine powder using a pestle. Powder was air dried and stored at 4°C.
To preabsorbed serum, 3 mg of insoluble cell powder was weighed for every microliter of serum. One ml of TBST solution (Tris-buffered saline, 1% Tween, pH 7.4) was added to every 3 mg of insoluble cell powder, cells were vortexed and incubated for 30 min at 70°C. Cells were vortexed again and spun down at 10,000 × g for 1 min. Cell pellets were washed with TBST until supernatant came clear after centrifugation. After the final wash, cell pellets were mixed with the rabbit serum, and incubated for 2 to 3 h at room temperature with gentle shaking. The serum and insoluble cell powder mix was spun down at 10,000 × g for 3 to 5 min, and pellets were discarded.
In vitro HCMV infections.
HFF cells were infected with an HCMV clinical isolate at different multiplicities of infection, from 0.5 to 5 PFU/ml. Cell monolayers were exposed to the virus for 1 hour, under normal growing conditions. After the incubation time, virus containing medium was removed, cells were washed with sterile phosphate-buffered saline, and medium was added.
Western blot analysis.
Different time points of HCMV-infected cells were collected by centrifugation and pellets resuspended in 0.01% sodium dodecyl sulfate solution. Protein concentrations were determined using Coomasie Protein Assay reagent (Pierce). Samples (5 μg of protein) were mixed with 2x SDS-polyacrylamide gel electrophoresis loading buffer (125 mM Tris [pH 7.0], 20% glycerol, 10% β-mercaptoethanol, 6% SDS, and 0.2% bromophenol blue), boiled for 5 min, and were then separated by PAGE on 12% acrylamide gels. In some cases duplicate gels were analyzed by Coomassie staining and Western blot.
For Western blot analysis, after semidry electrophoretic transfer of proteins onto polyvinylidene difluoride membranes (Westran S 0.2-μm pore; Schleicher & Schuell BioScience), the membranes were incubated overnight with 5% powdered milk in Tris-buffered saline-Tween buffer (150 mM NaCl, 10 mM Tris-HCl [pH 8.0], and 0.05% Tween 20) to block nonspecific protein interactions. The membranes were probed with one of the following primary antibodies: anti-LUNA preabsorbed rabbit serum (1:100 dilution), anti-IE1/2 monoclonal antibody (mAb810, Light Diagnostics), and anti-His coupled to horseradish peroxidase monoclonal antibodies (1:1,000; Invitrogen). Secondary antibodies used included goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated (Southern Biotechnology Associates, Inc.). Antibody-tagged protein bands on the probed membranes were detected using diaminobenzidine peroxidase substrate kit (Vector).
Infected cells were plated on glass coverslips, and fixed at different times postinfection with 3:1 methanol:acetone and then permeabilized using a combination of glycine and Triton X-100. First coverslips were treated with 10 mM glycine solution for 30 min at room temperature, after a DPBS rinse 30 min incubation with 0.1% Triton X-100 solution in phosphate-buffered saline was done at room temperature. The coverslips were rinsed three times in PBT (phosphate-buffered saline plus 0.5% Tween), and then incubated with primary antibody solution in phosphate-buffered saline for 90 min at room temperature.
After incubation, the coverslips were washed in PBT three times for 5 min each and then incubated with a 1:1,000 dilution in phosphate-buffered saline of an Alexa Fluor conjugated secondary antibody (Molecular Probes, Inc.) for 30 min in the dark at room temperature, and rinsed again with PBT. All antibody solutions also contained 2% fetal bovine serum. The cells were again washed in PBT three times for 5 min each and then were incubated with Hoechst 33258 staining solution for 5 min at room temperature, in the dark. Prior to the addition of mounting solution, the cells were rinsed with PBT. Primary antibodies included Mab810 (Light Diagnostics), mouse monoclonal antibody against HCMV IE1-2 proteins, and rabbit polyclonal anti-LUNA. Secondary antibodies used were Alexa Fluor 555-conjugated (red) goat anti-mouse and Alexa Fluor 488-conjugated (green) goat anti-rabbit immunoglobulin antibody (Molecular Probes, Inc.) Images were captured with a Nikon E800 epifluorescence/confocal microscope using a 20× objective, or a 100× oil immersion objective.
Accession numbers and alignments.
The following sequences were used for alignment analysis: gi
AD169-BAC isolate (nucleotides 102936 to 102538), gi
HEHCMVCG AD169 (nucleotides 117712 to 118110), gi
PH-BAC isolate (nucleotides 109303 to 108905), gi
Towne-BAC isolate (nucleotides 152184 to 152582), gi
Towne (nucleotides 117157 to 117555), gi
Merlin (nucleotides 119014 to 119412), gi
FIX-BAC isolate (nucleotides 109215 to 108817), gi
TR-BAC isolate (nucleotides 154705 to 155103), gi
Toledo-BAC isolate (nucleotides 104619 to 104218), and gi
chimpanzee cytomegalovirus (nucleotides 119078 to 119407). Protein alignment was done using MegaAlign, from Lasergene (DNAstar, Inc.), using Clustal W algorithms.