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Human cytomegalovirus sequences expressed in latently infected individuals promote a latent infection in vitro

Felicia Goodrum, Matthew Reeves, John Sinclair, Kevin High and Thomas Shenk

Article Figures & Data

Figures

  • Figure 1

    Low-passage clinical strains of HCMV, but not laboratory strains, establish a latent infection in cultured CD34+ hematopoietic progenitor cells. (A) Limiting dilution analysis was used to determine frequency of infectious center formation in CD34+ cells infected with low-passage (FIX and Toledo) or laboratory (Towne and AD169) strains at 10 to 14 dpi. The frequency is calculated as 1 over the number of cells required to yield one infectious center. The bars represent the average frequency of infectious center formation in the reactivation experiment (■) or the lysate control (▒) in at least 3 independent experiments for FIXwt, Toledo, and AD169 and two independent experiments for Towne. The standard error of the means is shown. The top number above each pair of bars reports the fold increase in infectious center formation in the reactivation compared with the lysate control. Using the paired Student t test, the probability (P) that the difference between the 2 averages is significant was calculated. Statistical significance is indicated by ** for P ≤ .01. All other pairs do not represent a significant difference (P > .05). The line marks the limit of detection (LD) for the assay. (B) CD34+ cells infected with Toledo or AD169 at a multiplicity of 5 PFU/cell were purified 20 hours later by FACS and seeded into long-term bone marrow cultures. Linearly amplified RNA was analyzed using the HCMV array at 20 dpi infection. All RNAs were hybridized in triplicate. Arabidopsis cDNA spots used as positive controls are circled. Cellular cDNA spots used for normalizing arrays are boxed. All other spots represent HCMV ORFs.

  • Figure 2

    Schematic of ULb′ region recombinant viruses. Large regions or individual ORFs within the ULb′ region were substituted by linear recombination into the FIX strain cloned as a BAC. Black bars represent the region missing in the recombinant virus, and the nucleotides missing are shown to the right of each variant.

  • Figure 3

    Growth kinetics of recombinant viruses. MRC5 cells were infected at a multiplicity of 0.05 PFU/cell with the FIXwt or each recombinant containing (A) a large substitution in the ULb′ region or (B) a substitution in an individual ORF within the FIX(ur)sub2 region. Lysates were collected over a time course and virus yields determined by TCID50 assays on MRC5 cells. The standard error is shown for 3 to 4 independent experiments.

  • Figure 4

    UL138 promotes a latent infection in cultured hematopoietic progenitor cells. (A) The latency phenotype of each virus in CD34+/CD38 cells was analyzed by limiting dilution assay. The bars represent the average frequency of infectious center formation in the reactivation experiment (black) or the lysate control (gray) in at least 3 independent experiments for FIXpar, FIX(ur)sub2, FIX(ur)sub3, and AD169. The top numbers above each pair of bars represent the fold increase in infectious center formation in the reactivation compared with the lysate control. The line indicates the limit of detection (LD) for the assay. (B) The latency phenotype was analyzed for single ORF substitution viruses by seeding 10 000 cells or an equivalent cell lysate per well into each of 24 wells. The bars represent the fraction of GFP+ wells in the reactivation experiment (■) or the lysate control (▒) in at least 3 independent experiments or 2 independent experiments for FIXsubUL141 and Towne. For panels A and B, statistical significance is indicated by * for P < .05 and ** for P ≤ .01 where there were 3 or more experiments. All other pairs do not represent a significant difference (P ≥ .05). The standard error of the means is shown.

  • Figure 5

    Detection of UL138 RNA in hematopoietic cells isolated from seropositive donors. (A) RNA isolated from CD34+ cells of seropositive (lanes 1-5) or seronegative (lanes 6-8) donors was analyzed by RT-PCR for (i) UL138, (iii) IE1, and (iv) GAPDH gene expression. RNA was amplified in panel ii with no prior RT. (B) RNA isolated from peripheral blood monocytes of seropositive (lanes 4, 6, 7, 9, and 11) or seronegative (lanes 1-3, 5, 8, 10, 12, 13) donors was analyzed by RT-PCR for (i) UL138, (iii) IE1, and (iv) GAPDH gene expression. RNA was also amplified in panel ii with no prior RT. (C) RNA isolated from CD34+ cells (lanes 1, 2, 5, 6) and CD34-derived dendritic cells (DDCs) (lanes 3, 4, 7, 8) of 2 further seropositive donors was analyzed by RT-PCR (lanes 1-4) for (i) UL138, (ii) IE1, and (iii) GAPDH gene expression. RNA was also amplified in an (i) UL138-PCR and (ii) with no prior RT (lanes 5-8). In all experiments, PCR products were transferred to a nitrocellulose membrane and probed with internal sequences to UL138 and IE1 genes.

Tables

  • Table 1

    Primers used for recombinant FIX virus construction

    NameHCMV ntSequence
    15kbsub1 fwd33979atg ctc aca ctc tat ctc ttc aca gcg aca tgt tgc ttc gtc tat gcg ctc agc gag tca gtg agc gag g
    15kbsub1 rev39422tac aac gga cgc gct cgc tgt ttc ggt ctc agg tca tct gca ttg act cgc agg cgg ccg cac tag tga t
    15kbsub2 fwd39395acc gaa aca gcg agc gcg tcc gtt gta cag ttg tat agc agc aca cgc ctc agc gag tca gtg agc gag g
    15kbsub2 rev45002ttt taa cgg cga cag tac agc tcc cgt taa aca tta gat taa tag acg ctc agg cgg ccg cac tag tga t
    15kbsub3 fwd44846aac aca tat act ccc cac gat cct cga ac acct tac agc ata tga gca aac agc gag tca gtg agc gag g
    15kbsub3 rev48104cga acg acg tgt gac gag gac gtg gtt tcc gca agc ctc tac cga cgc cgc agg cgg ccg cac tag tga t
    15kbsub4 fwd3397915kbsub1 fwd primer used
    15kbsub4 rev4910415kbsub4 rev primer used
    UL136fwd39491tca agg gcg tgg aga tgc cag aaa tga cgt ggg act tgg acg ttg gaa ata cca cgt cgt gga atg cc
    UL136rev40064tta ccc cgc cag cag cac ctc cgc cgg caa ccg cgt cgt cgt tgc tat cgt ccc atg tgc agg tgc tg
    UL137fwd40210ggt tga ggg ggc cgt tcc cgc gcg agt gct gta caa aag aga gag act gga cca cgt cgt gga atg cc
    UL137rev40285acg gtc ctc tgt ccg gat cta cgt ccc agt ctc tct ctt ttg tac agc act ccc atg tgc agg tgc tg
    UL138fwd40360ctg cta tct agc tta cca ttg gca cga cac ctt caa act ggt gcg cat gta cca cgt cgt gga atg cc
    UL138rev40798tca cgt gta ttc ttg atg ata atg tac cat ggc tac ggt ggt gaa ctg cgt ccc atg tgc agg tgc tg
    UL139fwd41361atg ctg tgg ata tta att tta ttt gca ctc gcc gca tcg gcg agt gaa aca cca cgt cgt gga atg cc
    UL139rev41768tca ccg agg cgg agg tgg aaa tga gcc gtc ctg tgg ggg agt gta cga cct ccc atg tgc agg tgc tg
    UL140fwd41950atg acc cca gct cag act aac gcc act acc acc gtg cac ccg cac gac aca cca cgt cgt gga atg cc
    UL140rev42525tca cag ggt ctg atg aag ctg cca aga gtc gtg gct gtg gcg cag cgc gtt ccc atg tgc agg tgc tg
    UL141fwd42564atg aga cag gtc gcg tac cgc cgg cga cgc gag agt tcc tgc gcg gtg cta cca cgt cgt gga atg cc
    UL141rev43840tca cct ctt cat ctt ttt aac acc ggg gta act atc gta agt cgg tag gct ccc atg tgc agg tgc tg
    UL142fwd43913atg cgg att gaa tgg gcg tgt tgg tta ttc gga tac ttt gtg tca tcc gta cca cgt cgt gga atg cc
    UL142rev44830tta ctg acc gcg cca tac ttc gta tac gaa cct aac cgg cgt aaa gtg ttt ccc atg tgc agg tgc tg
    • Bolded sequences represent the portion of the primer specific to the pKan/LacZ or pKanfrtFLAG plasmid. Other sequences represent the CMV gene-specific portion of the primer.

  • Table 2

    RT-PCR primers and probes

    Primer sequencesTm; MgCl2; product size, bp
    UL13855°C; 1.5 mM; 333
        5′-TGC GCA TGT TTC TGA GCT AC-3′
        5′-ACG GGT TTC AAC AGA TCG AC-3′
    UL138 DNA probe55°C; 1.5 mM; 150
        5′-GAG CTG TAC GGG GAG TAC GA-3′
        5′-AGC TGC ACT GGG AAG ACA CT-3′
    UL122/12355°C; 2.5 mM; 194
        5′-ATT CTT CGG CCA ACT CTG GA-3′
        5′-ACA CGA TGG AGT CCT CTG CC-3′
    UL122/123 DNA probe55°C; 2 mM; 201
        5′-CCC TGA TAA TCC TGA CGA GG-3′
        5′-CAT AGT CTG CAG GAA CGT CGT-3′
    GAPDH60°C; 3 mM; 238
        5′-GAG TCA ACG GAT TTG GTC GT-3′
        5′-TTG ATT TTG GAG GGA TCT CG-3′
    • Primers are paired, with one forward and one reverse for each.

    • Tm indicates temperature.