Article Figures & Data
Figures
- Fig. 1.
Structure of recombinant HCMV BAC DNAs. (a) Diagram of recombinant BAC DNAs of wt, RdlUL79+F, RdlUL87+F, and RdlUL95+F viruses. When the recombinant HCMV BAC DNA with mutation of the ORF was constructed, the FRT sequence was inserted into the middle of the ORF, and then the Kanr gene was excised by FLP-mediated recombination, leaving only 34 bp of the FRT sequence. (b) Diagram of recombinant BAC DNAs with an epitope fused to the UL95 and UL87 ORFs. When an HA epitope fused to the UL95 ORF was inserted, reverse selection was performed as described in Materials and Methods to remove the drug resistance cassette. To construct BACUL95N neo+St or BACUL95C neo+St, a marker cassette containing the RpsL gene, conferring increased sensitivity to streptomycin, and the neomycin resistance marker, providing kanamycin resistance, was inserted into the N or C terminus of the UL95 ORF, respectively. Intermediate BAC clones were isolated based on resistance to kanamycin. In a second round of homologous recombination, the entire marker cassette was replaced with the sequence containing an HA epitope by counterselection using an oligonucleotide, as described in Materials and Methods. To construct a recombinant BAC with a myc epitope fused to the C terminus of the UL87 ORF, the reverse selection was repeated after construction of BACHAUL95 or BACUL95HA as described in Materials and Methods. To construct a recombinant BAC with a Flag epitope fused to the N terminus of the UL79 ORF, the reverse selection was repeated after construction of BACHAUL95UL87myc or BACUL95HAUL87myc as described in Materials and Methods. (c to e) Growth curves of parental HCMV, RUL95HAUL87myc, RHAUL95UL87myc, RUL95HAUL87mycflagUL79, and RHAUL95UL87mycflagUL79 at an MOI of 3 (c and d) or 0.01 (e). Virus titers were determined by TCID50 assay as described in Materials and Methods.
- Fig. 2.
UL79, -87, and -95 are required for viral growth. HFF cells in the presence or absence of the complementary proteins were infected at an MOI of 3. Virus titers were determined by TCID50 assay as described in Materials and Methods. (a) Parental virus; (b) RdlUL79; (c) RdlUL87; (d) RdlUL95.
- Fig. 3.
UL79, -87, and -95 are not required for viral DNA replication. HFF cells in the presence or absence of the complementary protein were infected at an MOI of 3 (a to c) or 0.01 (d to f). Viral DNA was quantified by real-time PCR with gB primers and probe as described in Materials and Methods. Real-time PCR with 18S primers and probe were also performed to serve as an internal control. Data are averages for three independent experiments. (a and d) RdlUL79; (b and e) RdlUL87; (c and f) RdlUL95.
- Fig. 4.
MIE RNA levels are similar in HFF cells at a low MOI but not in HFF cells preexpressing UL79 and UL87. A real-time RT-PCR assay to detect MIE transcripts was performed after infection with the recombinant viruses at a low MOI in HFF cells in the presence or absence of the complementary viral proteins. Total RNA was harvested 1, 2, and 3 days after infection with RdlUL79 (a), RdlUL87 (b), or RdlUL95 (c) at an MOI of 0.01 and was assayed by MIE-specific primers and probe as described in Materials and Methods. The assay was performed in triplicate, and standard errors of the means were determined. HCMV RNAs were normalized to G6PD RNA. Preexpression of UL79 and UL87, but not that of UL95, affected MIE RNA levels at a low MOI.
- Fig. 5.
Northern blot analyses of late gene transcription after infection with recombinant viruses. HFF cells in the presence or absence of the complementary protein were infected with the mutant recombinant viruses at an MOI of 3. Cytoplasmic RNA was harvested at 1, 2, and 3 dpi in the presence or absence of PAA as described in Materials and Methods. Northern blots are shown for IE1 (left), UL75 (gH) (middle), and UL99 (pp28) (right). 28S and 18S rRNAs served as controls for equal amounts of RNA loading. Lanes in panel a: 1, 3, and 5, HFF cells; 2, 4, and 6, HFF cells expressing the UL79 fusion protein; 1 and 2, 1 dpi; 3 and 4, 2 dpi; 5 and 6, 3 dpi. Lanes in panels b and c: 1, 3, 5, and 7, HFF cells; 2, 4, 6, and 8, HFF cells expressing the UL87 (b) or UL95 (c) fusion protein; 1 and 2, 1 dpi; 3 and 4, 2 dpi; 5 and 6, 3 dpi (b) or 2 dpi in the presence of PAA (c); 7 and 8, 2 dpi in the presence of PAA (b) or 3 dpi (c). (a) RdlUL79-infected cells; (b) RdlUL87-infected cells; (c) RdlUL95-infected cells.
- Fig. 6.
RNase protection assay for UL44 transcripts after infection with recombinant viruses. Cytoplasmic RNAs were harvested 1, 2, and 3 days after infection with RdlUL79 (a), RdlUL87 (b), or RdlUL95 (c) at an MOI of 3. Twenty micrograms of RNA was hybridized to the 32P-labeled antisense UL44 promoter probe at 37°C overnight before digestion with RNase T1. The antisense UL44 probe contains a sequence upstream of the transcription start site in all UL44 transcripts. The protected RNA fragments were subjected to electrophoresis in denaturing 6% polyacrylamide gels. Lanes in panel a: 1, probe lacking RNase T1; 2, 4, and 6, HFF cells; 3, 5, and 7, HFF cells expressing the UL79 fusion protein; 2 and 3, 1 dpi; 4 and 5, 2 dpi; 6 and 7, 3 dpi. Arrow 1, 2, or 3 indicates the transcript initiating at start site 1, 2, or 3, respectively. Lanes in panels b and c: 1, 3, 5, 7, and 9, HFF cells; 2, 4, 6, 8, and 10, HFF cells expressing the UL87 (b) or UL95 (c) fusion protein; 1 and 2, 1 dpi; 3 and 4, 2 dpi; 5 and 6, 3 dpi (b) or 2 dpi in the presence of PAA (c); 7 and 8, 2 dpi in the presence of PAA (c) or 3 dpi (b).
- Fig. 7.
UL95-HA, UL87-myc, and Flag-UL79 fusion proteins are expressed at early times after infection. Viral DNA synthesis was inhibited with PAA or GCV. Western blot analysis was performed using an antibody against immediate-early proteins pIE86 and pIE72 (UL122 and -123), UL44, the HA, myc, or Flag epitope, or GAPDH at the indicated times after infection with RUL95HAUL87myc or RUL95HAUL97mycflagUL79 in the presence or absence of PAA or GCV as described in Materials and Methods. GAPDH served as a loading control. (a) Anti-HA antibody. (b) Anti-myc antibody. (c) Anti-Flag antibody. (d) Anti-IE72/IE86, anti-UL44, anti-p28, and anti-GAPDH antibodies.
- Fig. 8.
Localization of UL95-HA, UL87-myc, and Flag-UL79 fusion proteins, UL44 protein, and viral DNA after infection with recombinant viruses. The cells infected with RUL95HAUL87myc (a to e) or RUL95HAUL87mycflagUL79 (f to h) were harvested at 1 dpi (a and f), 2 dpi (b, d, e, and g), or 3 dpi (c and h). Cells were fixed and treated with antibody for microscopy or for FISH analysis as described in Materials and Methods. (a to c) Colocalization of UL95-HA protein with UL87-myc protein. (d) Colocalization of UL95-HA protein with UL44 protein. (e) Colocalization of viral DNA with UL44 protein. (f to h) Colocalization of Flag-UL79 protein with UL44 protein.
- Fig. 9.
Localization of UL95-HA, UL87-myc, or Flag-UL79 fusion protein after infection with recombinant viruses in the presence of PAA or GCV. Cells infected with RUL95HAUL87myc (a to f and i) or RUL95HAUL87mycflagUL79 (g and h) in the presence of PAA (a, c, e, g, and i) or GCV (b, d, f, and h) were treated at 2 dpi as described in Materials and Methods. (a and b) Colocalization of HCMV DNA and UL44 protein. (c and d) Colocalization of UL95-HA and UL44 proteins. (e and f) Colocalization of UL87-myc and UL44 proteins. (g and h) Colocalization of UL79-Flag and UL44 proteins. (i) Localization of UL87-myc and UL57 proteins.
Tables
- Table 1.
PCR primers and oligonucleotides for construction of plasmids and HCMV BAC DNAs for this study
Primer or oligonucleotide Sequence (5′–3′) XhoIUL79ORFF CCGCTCGAGATGATGGCCCGCGACGAAGAGAACCC HindIIIUL79ORFR CCCAAGCTTTCACGTCGTTAGCCAGCGTCGGCATAT XhoIUL95ORFF CCGCTCGAGATGATGGCGGCGGCGGTGGTGCGAGCGGAG HindIIIUL95ORFR CCCAAGCTTTTAGATTCAACGTGATGAGACCCGCGTCGTTCAGC XhoIUL87ORFF CCGCTCGAGATGGCCGGCGCTGCGCCGCGCCGCCTCGGCTGCGAC UL87(815)HindIIIR CAACGCCCAGCCAAAGCTTTTGCAGCTCCAGC UL87(815)HindIIIF GCTGGAGCTGCAAAAGCTTTGGCTGGGCGTTG HindIIIUL87ORFR CCCAAGCTTTTCGTGATGCAAACCGCGCTCGCGGCGACGTG BACdlUL79FRTFKanF GCGGCCGTGTTCGATGAAACGCGCGCCGCCCGTCTCAGCCAGCGCCTGTGTCACCCGCGCTTGAGCGGCGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAACTCAGCAAAAGTTCGATTTATTCAAC BACdlUL79FRTRKanR TTGCGCGCCAGGTCTTCGGGGAAAACGACCGGCAGGCCGGTGTGGCGCTGCACAAAGCGCGTCAGCAGTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTAATGCTCTGCCAGTGTTACAACCA BACdlUL87FRTFKanF GTCACTGCCGCGCGACCCGGCCGCAGATCGCGTTCGACGTTACGTATGCATTATCTCGCGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAACTCAGCAAAAGTTCGATTTATTCAAC BACdlUL87FRTRKanR GTCTCCGACCGCCGTTGACAGTGTTTACGCCATCTCTCCCCGTACCGAGCGTACATGAGAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTAATGCTCTGCCAGTGTTACAACCA BACdlUL95FRTFKanF ACGACGCCGCCACGCCGTCTTTTCTACGTCGACACGACGTGCTGGAGCGTTTCGCGGCCGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAACTCAGCAAAAGTTCGATTTATTCAAC BACdlUL95FRTRKanR CACGGTCAGCATTGCGTAACGCATAATCACGCACACAAAGCGACGGCAAAGGCTCAGCCGGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTAATGCTCTGCCAGTGTTACAACCA BACUL95Nneo+StF CTTCTGCAGCTCCGCGTAGCGCTCCTGGATCTTGGCGGCCGAGTCTCCGCGCAACGGCCTGGTGATGATGGCGGGATC BACUL95Nneo+StR TTTTCCTCTCCTCTCGCCGCTGCCGCCTAACCTCCGCTCGCACCACCGCCGCCGCTCAGAAGAACTCGTCAAGAAGG BACUL95Cneo+StF CGGAAAGTTGCTGGACGCCCTCTCGCTGAACGACGCGGGTCTCATCACGTTGAATGGCCTGGTGATGATGGCGGGATC BACUL95Cneo+StR TCAAGGTCGACGCGCATCACGTCCTTTAAGAGCTGTTTGTTGACCGACGTCATAGTCAGAAGAACTCGTCAAGAAGG BAColigoHAUL95 TTGCGCTTCTGCAGCTCCGCGTAGCGCTCCTGGATCTTGGCGGCCGAGTCTCCGCGCAACATGTACCCATACGATGTTCCAGATTACGCTGCGGCGGCGGTGGTGCGAGCGGAGGTTAGGCGGCAGCGGCGAGAGGAGAGGAAAAAGATG BACUL95Coligo CCACGTCGGAAAGTTGCTGGACGCCCTCTCGCTGAACGACGCGGGTCTCATCACGTTGAATTACCCATACGATGTTCCAGATTACGCTCTATGACGTCGGTCAACAAACAGCTCTTAAAGGACGTGATGCGCGTCGACCTTGAGCGACA BACUL87Cneo+StF CAGCGTTGACGGCAGTTCTGAACCCACGTCGCCGCGAGCGCGGTTTGCATCACGAGGCCTGGTGATGATGGCGGGATC BACUL87Cneo+StR GTCGGACGCTCCTCCGGACGAAACGCCGCGGCGGCAGCGGCCGCGGCTTCCATCATCAGAAGAACTCGTCAAGAAGG BACUL87oligo TCCGTCAGCGTTGACGGCAGTTCTGAACCCACGTCGCCGCGAGCGCGGTTTGCATCACGAGAACAGAAACTGATCTCTGAAGAAGACCTGTGATGGAAGCCGCGGCCGCTGCCGCCGCGGCGTTTCGTCCGGAGGAGCGTCCGACGCCGG BACUL79NFneo CACAGCCTGCGGCTGCTGCTCGTCCATCGTCATTGTCGTCACCGTCGCTACCCGCTCACCGAGCGAACGGGCCTGGTGATGATGGCGGGATC BACUL79NRneo GATTGGCGCAAGTAAAGGAGAATTTGCCTGTGCGGACCCGCGGGACGGCGGGGTTCTCTTCGTCGCGGGCTCAGAAGAACTCGTCAAGAAGG BAColigoflagUL79 GCGGCTGCTGCTCGTCCATCGTCATTGTCGTCACCGTCGCTACCCGCTCACCGAGCGAACGATGGACTACAAAGACGATGACGACAAGGCCCGCGACGAAGAGAACCCCGCCGTCCCGCGGGTCCGCACaGGCAAATTCTCCTTTACTTG UL79detectF CGCCAGGCCTTCCCGGGGCTGG UL79detectR CCGTAGAGCGTGCCTAGGGAGAAGAGG UL87BACdetectF CGATCCGTAAGCCGCGGTTACGG UL87BACdetectR CGTAAAGTCAAATGGCGGCGTGGCG UL95BACdetectF GGGAAAAGAACGGCGGTGGGTCTCG UL95BACdetectR GCTGGAGAGTGACAGCTCCACGTGAGC UL95NdetectF AGTTCGGGGTGCATCATCT UL95NdetectR ACGACACCACTAGGGACGAC UL95CdetectF AAGTTGCTGGACGCCCTCTC UL95CdetectR ACACGCATCACCGTCAAAG UL87CdetectF CCCATTGTGTGTGTCCTCAT UL87CdetectR CCGTACCGTCGTCCATTAAC UL79NdetectN GGTCGTAACGGGCGAGAAAGCCG UL79NdetectR CTGTCCGCGCGACATCTTCTCGC
