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. 1997 Dec 9;94(25):13985-90.
doi: 10.1073/pnas.94.25.13985.

Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs

H Zhu  1 , J P Cong, T Shenk
Affiliations

Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs

H Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

We used differential display analysis to identify mRNAs that accumulate to enhanced levels in human cytomegalovirus-infected cells as compared with mock-infected cells. RNAs were compared at 8 hr after infection of primary human fibroblasts. Fifty-seven partial cDNA clones were isolated, representing about 26 differentially expressed mRNAs. Eleven of the mRNAs were virus-coded, and 15 were of cellular origin. Six of the partial cDNA sequences have not been reported previously. All of the cellular mRNAs identified in the screen are induced by interferon alpha. The induction in virus-infected cells, however, does not involve the action of interferon or other small signaling molecules. Neutralizing antibodies that block virus infection also block the induction. These RNAs accumulate after infection with virus that has been inactivated by treatment with UV light, indicating that the inducer is present in virions. We conclude that human cytomegalovirus induces interferon-responsive mRNAs.

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Figures

Figure 1
Figure 1
Characterization of UV HCMV. (A) Western blot showing that UV irradiation of virions blocks expression of the HCMV IE1 and IE2 RNAs, but has no effect on the delivery of a virion protein to cells. HF cells were mock-infected or infected, and extracts were prepared 8 or 21 hr later. Lanes 1–6 were reacted with antibody that binds to IE1 and IE2, whereas lanes 7–9 were reacted with antibody to pp65. The molecular weights of marker proteins are indicated on the left. (B) Northern blot showing that IE1 RNA is detected at 8 hr after infection of HF cells with HCMV but not after infection with UV HCMV. (C) Immunofluorescent localization of pp65 and IE1/2 within infected cells. HF cells were infected with HCMV or UV HCMV for 2 or 8 hr, reacted with antibody to pp65 or IE1/IE2 followed by a fluorescein-labeled secondary antibody (bright nuclear signal), and counterstained with ethidium homodimer-1 (dim whole cell signal).
Figure 5
Figure 5
Kinetic analysis of cig RNA accumulation. HF cells were mock-infected (M) or treated with the inducers identified to the right (HCMV, HSV-1, and IFN-α), RNA was prepared at various times after treatment (indicated above lanes), and analyzed by Northern blot by using the probes indicated on the left (cig1, cig49, HCMV IE1, and HSV-1 icp47).
Figure 2
Figure 2
Differential expression of RNAs in HF cells assayed by Northern blot. (A) RNA was prepared from mock-infected (M), HCMV-infected cells (C), or UV HCMV-infected cells (UV) and assayed by using cloned cDNA segments. The different clones (cigs) are identified above the panels. (B) RNA was prepared from mock-infected (M), HCMV-infected (C), or HCMV-infected cells that were treated with cycloheximide (CX) and assayed as in A. (C) RNA was prepared from mock-infected cells (M), HCMV-infected cells (C), or cells treated with interferon α (I) and assayed by using probes corresponding to the cigs or the mxA gene. (D) RNA was prepared as in A and assayed with probes corresponding to interferon-inducible genes (mxA, isg15K, and IFN-β) or control genes that are not induced by interferon (p53, p21, cPLA2, and actin).
Figure 3
Figure 3
The HCMV particle mediates the induction of differentially expressed HF RNAs. (A) Requirements for induction monitored by Northern blot assay. The relative amounts of three cellular RNAs (cig1, cig6, and cig49) were monitored in mock-infected cells (mock), HCMV AD169-infected cells (HCMV), cells treated with medium from which virions were removed by filtration (inf. med.), mock-infected cells treated with cycloheximide (CX), HCMV AD169-infected cells treated with cycloheximide (CX+HCMV), cells infected with purified HCMV AD169 particles (virions), cells infected with purified noninfectious enveloped particles from HCMV AD169 (NIEPs), adenovirus-infected cells (Ad dl309), herpes simplex type 1-infected cells (HSV-1), HCMV Towne-infected cells, HCMV Toledo-infected cells, interferon α-treated cells (IFN-α), and cells treated with interferon that was added to medium and passed through the filter type used to exclude virus (fil. IFN-α). (B) Northern blot assay showing that antibody that neutralizes HCMV (antibody C) blocks the induction of cig RNA accumulation, whereas antibodies that neutralize interferon α or β (antibody Iα, Iβ) block the induction of cig RNAs by interferon α or β (inducer Iα, Iβ) but have no effect on the induction of cig RNAs by HCMV (C). RNA prepared from mock-infected control cells is designated M. The cellular cytosolic phospholipase A2 RNA was assayed as a loading control (control, cPLA2).
Figure 4
Figure 4
Requirements for the induction of cig RNA accumulation. (A) An intact HCMV particle is required. Purified virions were treated with a mixture of Triton X-100 and deoxycholate (T/C) and separated by centrifugation into supernatant (S) and pellet (P) fractions. Northern blot assays show the effect of detergent treatment on the induction of two cig RNAs (cig1 and cig49) by virions (HCMV) or interferon α (IFN-α). (B) The induction of cig RNAs does not involve the release of mediators stored within infected HF cells. At 8 hr after treatment, RNA was prepared from mock-infected cells (lane 1), HCMV-infected cells (lane 2), or a 9:1 mixture of mock and infected cells (lanes 3 and 4). The two mixed cultures differed in the time of mixing. In lane 3, cells were mixed at 1 hr after infection; in lane 4, RNAs were prepared and mixed from 8 hr mock- and HCMV-infected cells. RNAs were analyzed by Northern blot by using cellular (cig1, cig6, cig49, and cPLA2) and viral (IE1) probes.
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