Article Figures & Data
Figures
- Fig. 1.
Recombinant virus construction and structure. (A) The top line shows an EcoRI restriction map of the CMV (Towne) genome, with the eight cosmids and the plasmids pON303G and pON2710 used to generate recombinant CMV depicted below. The mutation in pON2710 is marked “X.” The expanded pON303G/pON2710 region shows the SalI fragment of the plasmid endpoints and overlap with adjacent cosmids (Tn15 and Tn51), including relevant restriction sites, and the probe (probe 303 NsiI; filled bar) used in the analysis. The bottom shows nucleotide sequence from the CAAT box (underlined) to PSS sequences of the wild-type (pON303G) and mutant (pON2710), with theRsrII site introduced at nt 173786 of the viral genome into pON2710 shown in italics, the pORF94 start codon ATG or mutant ATC in boldface, and the introduced stop codon underlined. (B) Autoradiogram of EcoRI (left)- or EcoRI plus RsrII (right)-digested viral DNA from parental CMV (Towne), two independent isolates of cosmid-derived Towne (RC303.1 and RC303.2), and two independent isolates of cosmid-derived pORF94 mutant (RC2710.1 and RC2710.2), hybridized with the 303 NsiI probe that had been gel purified and random primed with digoxigenin (Boehringer Mannheim) (19). Size markers are indicated to the left.
- Fig. 2.
Growth curves of RC303.1 (filled squares) and RC2710.1 (open squares) following infection of HFFs at an MOI of 0.02 (input virus was plotted at day 0). Cells and media were collected at the indicated time points, and virus yield was measured in duplicate by plaque assay with both values falling within the symbol shown. The detection limit (horizontal line) was 10 PFU/ml.
- Fig. 3.
RNase protection of PSS and LSS in HFFs. (A) PhosphorImage depicting RNase protection of PSS-initiated transcripts in total cellular RNA at 8 hpi at an MOI of 3 (upper panel). The panel shows uninfected cell RNA (Mock), three lanes with CMV-infected cell RNA (Towne, RC303, and RC2710), yeast control RNA (Yeast), and the undigested probe (Probe). Total RNA (5 μg) was incubated with riboprobe complementary to sequences from nt −218 to +120 relative to PSS. On the right, the 420-nt undigested probe (Probe), 338-nt fully protected band (Full protection), 156- to 172-nt species generated by the presence of the mutations (Mutation mismatch), and the 120-nt PSS protected species (PSS) are shown. Actin control RNase protection (lower panel) with a 334-nt probe (Probe) generated a 245-nt protected band (Actin) used as a loading control. (B) PhosphorImage depicting RNase protection of LSS- and PSS-initiated transcripts in total cellular RNA at 48 hpi at an MOI of 1 (upper panel). The panel shows yeast control RNA (Yeast), uninfected cell RNA (Mock), three lanes with CMV-infected cell RNA (Towne, RC303, and RC2710), and the undigested probe (Probe). Total RNA (20 μg) was incubated with riboprobe complementary to sequences from nt −453 to +120 relative to PSS. On the right, the 694-nt undigested probe (Probe), 476-nt LSS1 protected band (LSS1), 299-nt (5′ end) and 172-nt (3′ end) LSS1 protected bands resulting from digestion at the mutation sites, and the 120-nt PSS protected species (PSS) are shown. Actin control RNase protection (lower panel) with a 334-nt probe (Probe) generated a 245-nt band (Actin) used as a loading control. [γ-32P]ATP-end-labeled 100-bp ladder (GibcoBRL) was used as a size marker; intensities of bands were determined with the ImageQuant version 2.0 software (Molecular Dynamics), and volume quantitation reports were analyzed at exposures below saturation.
- Fig. 4.
Detection of sense CLTs by RT-PCR in GM-Ps. (A) Ethidium bromide-stained agarose gel following separation of PCR products amplified by IEP3D and IEP1K for 30 cycles and nested for 30 cycles using IEP2D and IPE1G. (B) Autoradiogram of DNA blot hybridization with α-32P-end-labeled IEP1H after transfer to a nylon membrane. Lanes (left to right): 100-bp ladder, RC303 and RC2710 amplified with (complete) or without (no SSII) the addition of Superscript II, and a control containing no added cDNA (No cDNA). The spliced cDNA is amplified as a 206-bp product (arrow), and the DNA is amplified as a 1,032-bp product.
Tables
- Table 2.
QC-PCRa
GM-P culture no. Day postinfection CMV genome copy no./cellb RC303 RC2710 20c 18 0.2–0.8 0.2–0.8 22c 17 2 2 25d 19 1–3 0.8–2 40d 16 1–4 1–4 45d 15 8 20 46d 18 8 8 ↵a GM-Ps latently infected separately with RC303 and RC2710 were analyzed for CMV DNA content by QC-PCR.
↵b Determined by competition with threefold dilutions of competitor DNA; when competition between the competitor and genomic DNA spanned two dilutions, the data are represented as a range.
↵c RC303.2 and RC2710.2 were used.
↵d RC303.1 and RC2710.1 were used.
