Abstract

Human Cytomegalovirus (CMV) recombinants were isolated from its natural host, Peromyscus maniculatus, and named Peromyscus CMV (PCMV). A recombinant PCMV was constructed containing Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame with the enhanced green fluorescent protein (EGFP) gene inserted at a site homologous to the human CMV UL33 (P33) gene. Recombinant CMV was used for the expression and immunization of deer mice against SNV-G1.

The results of the study indicate that P. maniculatus could be infected with as few as 10 viral particles of recombinant virus. Challenge of P. maniculatus with recombinant or wild-type PCMV did not produce any obvious pathology in infected animals. P. maniculatus immunized with the recombinant virus developed an antibody response against SNV and EGFP. When rechallenged with the recombinant virus, the animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.

Culture of cells and viruses.

PCMV was cultured on P. manipulates embryonic cells (PMEC). Virus stocks were prepared by infecting PMEC spinner flasks in Iscove’s Modified Dulbecco’s Medium (IMDM) with 2% fetal calf serum (FCS) at a multiplicity of infection (MOI) between 0.05 and 0.1 at 37 °C When 100% of the cells showed cytopathology but were still adherent, the cells were scraped and the suspension clarified by pelleting in a Sorvall GLC centrifuge at 1500 × g for 5 min. Cell pellets were resuspended in 10 ml IMDM with 2% FCS and subjected to three cycles of freezing and thawing. The cell lysate was clarified by sedimentation in a Sorvall GLC centrifuge at 1500 × g for 5 min.

The virus titer was determined by inoculating PMEC into 24-well plates with a serial dilution of virus stock in IMDM-2% FCS at 100 µl/well for 1 hour at 37°C. The 24-well plates were cultured with an overlay containing IMDM with 0.5% agarose, 2% FCS and 50 µg kanamycin/ml for 1 week, after which the plates were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 1 h, the overlay was removed, and the cells were stained with 0.6% methylene blue. After a water wash, the plates were blotted dry and the plates were counted.

Preparation of PCMV genomic clones.

The PCMV virus stock was prepared as described above, after which the PCMV was pelleted at 15,000 rpm in an SW27 rotor (Beckman) for 1 hour at room temperature. PCMV genomic DNA was extracted with the PureGene DNA Extraction Kit (Gentra Systems) according to the manufacturer’s protocol. Virus genomic DNA was digested with NotI for 2 h and the reaction was stopped by heating at 65 °C for 20 min.

Restricted virus DNA was ligated into pET29c (Novagen) which was linearized with NotI and treated with shrimp alkaline phosphatase (USB). Electrocompetent Escherichia coli DH10B (Life Technologies) was transformed with the ligation mixture at 330 μF and 400 V (with the voltage amplifier set to 4 kΩ). Clones were selected with ≥10 kb NotI inserts. The viral origin of the NotI inserts was verified by Southern blotting (not shown).

RT-PCR of the P33 gene.

Total RNA from PCMV-infected PMEC was extracted using Trizol (Life Technologies) according to the protocol provided by the manufacturer. RNA was reverse transcribed with the Thermoscript Reverse Transcription-PCR (RT-PCR) system (Life Technologies) using the random hexamers supplied with the kit. The resulting cDNA was treated with DNase I (Promega).

The 5′ end of the gene was subsequently amplified by PCR with the forward primer 5′-CATACACAATAAAAGCCTCCAGAT-3′ and the reverse primer 5′-CGTCCGGTAGTACATAAGTGC-3′, which are located on either side of the putative splice site. The same primers were used in a PCR to amplify part of the P33 gene using genomic DNA from the virus as a template. The PCR product, representing the P33 cDNA sequence, was cloned into pGEM-T Easy according to the protocol (Promega). The PCR product was sequenced to confirm the absence of intron sequences.

Immunohistochemistry.

PMCs were plated on coverslips in six-well plates and infected with wt-PCMV or recombinant PCMV at a virus/cell ratio (MOI) of 0.1. At 6 days post-infection, cells on coverslips were fixed by incubation in methanol-acetone (3:1) solution for 5 min. The coverslips were then dried and stored at -20°C. Before immunostaining, coverslips were rehydrated in PBS for 5 min, followed by incubation with primary antibody (1:200 dilution, recombinant human anti-SNV-G1 antibody, SNV immunoglobulin G1 #26) in PBS-Tween 20 at 0.2% for 1 h.

Cells were then washed three times for 5 min with PBS-0.2% Tween 20, incubated with secondary antibody (1:2000 dilution; goat anti-human immunoglobulin G antibody [H+L] conjugated with phosphatase alkaline; Vector Laboratories), washed three times for 5 minutes with PBS-0.2% Tween 20, rinsed with Tween 20-Tris buffered saline, and developed using Alkaline Phosphatase Substrate Kit 1 (Vector Laboratories ). Coverslips were mounted on slides and viewed using an interference contrast microscope.

SDS-polyacrylamide gel electrophoresis and Western Blot.

PMCs, plated in 60 mm dishes, were infected with PCMV wt or PCMV (ΔP33:G1EGFP) or mock-infected. Alternatively, PMCs were transfected by electroporation with pEGFP-N2-G1 as described above. For infected cells, cells were lysed after 5 days. For transfected cells, 1-day post-transfection, PMECs were superinfected with wt-PCMV at an MOI of 1.

After 6 days, lysates were obtained by aspirating medium and scraping cells into 100 μl sample buffer. 4x containing 0.4 M Tris-HCl (pH 6.9), 40% glycerol, 10% SDS, 0.016% BPB protease inhibitors (Boehringer Mannheim), and 10% β-mercaptoethanol, added just before its use. Cell lysates were heated at 100 °C for 5 min before being stored at -20 °C or used immediately.