In Situ PCR for the Detection of Human Cytomegalovirus in Suspension Cells During the Latent Phase of Infection

Barry Slobedman Summary

Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of latent viral DNA and transcripts in these cells was investigated using methods based on polymerase chain reaction (PCR)-driven in situ hybridization (ISH) and reverse transcription (RT)-PCR-driven ISH. Using a conventional thermal cycler, latent viral DNA or transcripts were amplified within suspension cells. Amplified products were then detected by nonisotopic ISH on cells cytospun on glass microscope slides. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in more than 90% of cells. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor mobilized peripheral blood or bone marrow from healthy seropositive donors. When evaluated by RT-PCR-ISH, only a small proportion of experimentally infected cells (approx 2%) had detectable latent transcripts. The application of PCR-ISH and RT-PCR-ISH has enabled the identification of the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode latent transcripts.

Key Words: Herpesvirus; human cytomegalovirus; latent infection; myeloid progenitor cell; suspension cells; in situ PCR; in situ RT-PCR; viral DNA; viral transcripts.

1. Introduction

Human cytomegalovirus (CMV) is a medically important ^-herpesvirus carried by a majority of individuals, in whom it is a leading cause of opportunistic and congenital disease (1). Like other herpesviruses, primary infection by CMV leads to a life-long latency that is characterized by the maintenance of the viral genome without active infectious virus production. Periodically throughout life, virus reactivates from latency and is shed in bodily secretions, including saliva, urine, and breast milk. Although primary and reactivated infection

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

remain largely asymptomatic in immunocompetent individuals, primary infection by CMV is a significant cause of serious congenital disease, leading to neurological damage in children. Reactivated infection is a major cause of disease in immunocompromised individuals, including patients with AIDS and allograft transplant recipients (1).

Peripheral blood (PB) and bone marrow (BM)-derived monocytes and granulocyte-macrophage progenitor cells (GM-P) are sites of CMV latency. The viral genome is maintained in cultured primary GM-Ps during experimental latent infection (2-5) and is detected in naturally infected PB- and BM-derived mononuclear cells from healthy, seropositive donors (6-9). Although the CMV genome is transcriptionally repressed during latent infection, a number of CMV latency-associated transcripts have been detected during latency (4,5,10). The ability of this virus to reactivate from a latent state contributes significantly to its success as a human pathogen, yet the tissue distribution of latent CMV has been poorly understood (11).

Quantitation of latent infection during natural infection has proved complicated primarily because of the need to apply polymerase chain reaction (PCR) methods to detect latent viral DNA and transcripts. Methods that rely on in situ hybridization (ISH) to enumerate RNA- or DNA-positive cells, particularly when combined with PCR amplification to increase sensitivity for low copy numbers, provide an accurate picture of the distribution of viral nucleic acids in host cells and tissues, although direct (i.e., non-PCR based) in situ detection of CMV DNA in latently infected cells has been reported (12).

This report describes the adaptation of PCR-driven ISH (PCR-ISH) and reverse transcription (RT)-PCR-driven ISH (RT-PCR-ISH) methods (13,14) to enumerate suspension cells harboring latent CMV genomes during experimental (Fig. 1) and natural latent infection (Fig. 2) or to enumerate latently infected cells expressing a class of CMV latency-associated transcripts from the major immediate early (IE1/IE2) region of the viral genome (Fig. 3 [15]). The procedure can be divided into two main steps: (1) in situ amplification of latent viral DNA or RNA, which is performed on cells in suspension using a conventional thermal cycler; and (2) the detection of amplified products within cells by nonisotopic ISH, which is performed on cells after being cytospun on glass microscope slides.

2. Materials

2.1. Preparation of Cells

1. Ficoll (Lymphoprep or similar).

2. HEPES-buffered saline solution (HBSS).

4. 100 mMKHCO3 stock.

Fig. 1. Photomicrographs showing detection of CMV DNA in experimentally infected GM-P culture by PCR-ISH. Cells from CMV-infected (A-C) or mock-infected (D) cultures were subjected to PCR-ISH, except that Taq DNA polymerase was omitted from C. Filled arrow, CMV DNA-positive cell; and open arrow, CMV DNA-negative cell. Magnification is A, x1280 and B-D; x3200. (E) To confirm the specificity of the in situ PCR amplification of CMV DNA, total DNA was extracted from CMV-infected (Inf, complete) or mock-infected GM-Ps (Mock, complete) after PCR amplification with IEP3A and IEP3B or with the omission of Taq DNA polymerase (Inf, - Taq and Mock, -Taq) and subjected to DNA blot hybridization. Filter was probed with a 32P-labeled probe derived from pON2810, washed, and exposed to X-ray film for 2 h. The predicted CMV-specific PCR product size of 167 base pairs is indicated.

Fig. 1. Photomicrographs showing detection of CMV DNA in experimentally infected GM-P culture by PCR-ISH. Cells from CMV-infected (A-C) or mock-infected (D) cultures were subjected to PCR-ISH, except that Taq DNA polymerase was omitted from C. Filled arrow, CMV DNA-positive cell; and open arrow, CMV DNA-negative cell. Magnification is A, x1280 and B-D; x3200. (E) To confirm the specificity of the in situ PCR amplification of CMV DNA, total DNA was extracted from CMV-infected (Inf, complete) or mock-infected GM-Ps (Mock, complete) after PCR amplification with IEP3A and IEP3B or with the omission of Taq DNA polymerase (Inf, - Taq and Mock, -Taq) and subjected to DNA blot hybridization. Filter was probed with a 32P-labeled probe derived from pON2810, washed, and exposed to X-ray film for 2 h. The predicted CMV-specific PCR product size of 167 base pairs is indicated.

2.2. In Situ PCR Amplification

All molecular biology reagents are from Invitrogen and Roche.

1. Phosphate-buffered saline (PBS).

2. Paraformaldehyde solution: 4% made in PBS.

3. 10X PCR buffer (Invitrogen).

4. 100 mM dATP stock.

5. 100 mM dCTP stock.

6. 100 mM dGTP stock.

7. 100 mM dTTP stock.

8. Gelatin: 1% solution.

9. RNase- and DNase-free water.

10. Mineral oil.

11. TaqDNA polymerase.

12. Glutaraldehyde-activated 3-aminopropyl-triethoxysilane (APES)-coated glass microscope slides (or similar coated slides compatible with ISH).

Donor: 12 3^5670 9 Id 11 Ii 1 2 3 4 S E 7 i «tStltl

Complete -Taq

Fig. 2. (A) Photomicrograph showing detection of CMV DNA in naturally infected mononuclear cells by PCR-ISH. CMV DNA-positive cells are indicated by an arrow. Magnification is x3200. (B) PCR-ISH determination of the percentage of CMV DNApositive mononuclear cells from 12 separate granculocyte colony-stimulating factor-mobilized PB or BM donors. Cells were analyzed in the presence of the full complement of reagents for CMV DNA detection (complete) or with the omission of Taq DNA polymerase (- Taq).

Donor: 12 3^5670 9 Id 11 Ii 1 2 3 4 S E 7 i «tStltl

Complete -Taq

Fig. 2. (A) Photomicrograph showing detection of CMV DNA in naturally infected mononuclear cells by PCR-ISH. CMV DNA-positive cells are indicated by an arrow. Magnification is x3200. (B) PCR-ISH determination of the percentage of CMV DNApositive mononuclear cells from 12 separate granculocyte colony-stimulating factor-mobilized PB or BM donors. Cells were analyzed in the presence of the full complement of reagents for CMV DNA detection (complete) or with the omission of Taq DNA polymerase (- Taq).

2.3. In Situ Detection of Amplified Products

All molecular biology reagents are from Invitrogen and Roche.

2. 1% Glutaraldehyde solution.

4. Acetylation medium: 0.25% acetic anhydride in 0.1 Mtriethanolamine, pH 8.0.

5. Deionized formamide.

6. Yeast tRNA: 10 mg/mL stock.

7. Sonicated salmon sperm DNA: 10 mg/mL stock.

8. 5X Hybridization buffer.

9. Dithiothreitol (DTT): 100 mM stock.

10. RNase inhibitor: 10 U/mL stock.

13. Rubber cement.

15. Washing solution: 0.1X SSC, 10 mMTris-HCl, pH 7.5, 30% deionized formamide.

16. Buffer 1: 100 mM maleic acid, 150 mMNaCl. Adjust to pH 7.5 with NaOH.

17. Blocking reagent for nucleic acid hybridization (Roche).

Fig. 3. Photomicrographs showing detection of CMV latency-associated transcripts (CLTs) from the IE1/IE2 region of the viral genome by RT-PCR-ISH in experimentally infected GM-P culture. Infected cells were subjected to RT-PCR-ISH (A and B), with controls omitting reverse transcriptase (C) or Taq DNA polymerase (D). CLT-posi-tive cells are arrowed. Magnification is A; x1280 and B-D; x3200. (E) RT-PCR-ISH determination of the percentage of CLT-positive cells from three separate GM-P cultures 2 to 3 wk after infection with CMV at a multiplicity of infection of 3. Virus (Inf) or mock (Mock) infected cells were analyzed in the presence of the full complement of reagents for sense CLT detection (complete) or with the omission of either reverse transcriptase (-RTase) or Taq DNA polymerase (-Taq).

18. Buffer 2 (prepare fresh): 1% (w/v) blocking reagent for nucleic acid hybridization in buffer 1.

19. Buffer 3: 100 mMTris-HCl, pH 9.5, 100 mMNaCl, 50 mMMgCl2.

20. Dimethylformamide: 100% and 70%.

21. Developing solution (30 mL): 135 pL of nitroblue tetrazolium chloride (NBT) stock (stock is 100 mg of NBT in 1.3 mL of 70% dimethylformamide), 105 pL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) stock. Stock is 50 mg of BCIP in 1.0 mL 100% dimethylformamide (see Note 1). Made up to 30 mL with Buffer 3.

2.4. In Situ Amplification and Detection of RNA Transcripts

The following are needed in addition to those reagents listed previously.

1. DNase digestion mixture (200 pL):

f. 177 pL of RNase- and DNase-free water.

g. 5 |L of 5X RT buffer (as supplied with the reverse transcriptase).

h. 5 |L of RT primer (IEP2D, 50 pmol/|L; see Note 2), i. 147 |L of RNase- and DNase-free water.

3. Reverse transcriptase (SuperScript II, Invitrogen).

3. Methods

3.1. Source of Cells

Detection of latent CMV infection was conducted using either (1) cultured primary human GM-Ps that had been latently infected in the laboratory with CMV (i.e., experimental latent infection) or (2) granulocyte colony-stimulating factor-mobilized PB or BM aspirates that had been collected from healthy donors (i.e., natural latent infection). BM or mobilized PB samples were layered over 15 mL of Ficoll (Lymphoprep or similar) and centrifuged for 15 min at 1000^. Cells were washed once in HBSS; treated with 155 mM NH4Cl, 10 mM KHCO3, pH 7.0, for 5 min to lyse any remaining red blood cells; and washed three times with HBSS before being processed further for PCR-ISH.

3.2. In Situ Amplification and Detection of Latent CMV DNA in Myeloid Progenitor Cell Suspensions by PCR-ISH

3.2.1. In Situ PCR Amplification

1. Wash cells (see Note 3) three times in PBS (see Note 4).

2. Resuspend cells in 4% paraformaldehyde (see Note 5) and fix for 30 min at room temperature.

3. Wash cells 3X in PBS (see Note 6). For the final wash, transfer cells to 0.5 mL of PCR tubes, and split cells as appropriate to include a -Taq DNA polymerase control.

4. Resuspend cells in 48 |L of PCR mixture to make 192 |L (enough for four reactions):

g. 4 |L of forward primer (IEP3A, 50 pmol/mL; see Note 7).

h. 4 |L of reverse primer (IEP3B, 50 pmol/mL; see Note 7).

i. 146 |L of RNase- and DNase-free water.

5. Overlay with mineral oil and place into a conventional PCR thermocycler.

6. Denature for 8 min at 94°C, with 2 |L of Taq DNA polymerase added after the first 4 min (see Note 8). Then continue the denaturation for 4 min before thermocyling as follows for 30 cycles:

b. 58°C, 2 min (as appropriate for gene specific primers).

8. Wash cells 1X in PBS. Resuspend in PBS.

9. Cytospin onto glass microscope slides coated for ISH applications, for example, glutaraldehyde-activated APES-coated slides (16).

3.2.2. In Situ Detection of Amplified Products

1. Fix the cells in 4% paraformaldehyde (see Note 5) for 30 min and then wash twice for 5 min in PBS.

The next two steps are only required if storing the slides at this point is desired. If continuing straight on with the procedure, omit the following two steps:

2. Wash twice in 50% ethanol (ETOH), once in 70% ETOH, once in 100% ETOH (5 min each wash), and air-dry (see Note 9). Slides can be stored at this point.

3. Rehydrate through graded ETOHs (100%, 70%, 50%) to PBS.

4. Fix 0.1% glutaraldehyde (in PBS) for 30 min at 4°C (see Note 10).

5. Wash twice for 5 min in PBS.

6. Permeabilize cells in 1% Triton X-100 (in PBS) for 2 min.

7. Wash four times briefly in PBS.

9. Acetylate in acetylation medium for 10 min (see Note 11).

10. Wash twice for 5 min in PBS.

11. Dehydrate through graded ETOHs (50%, 70%, 100%). Air-dry (see Note 12).

12. Prepare hybridization mixture in an RNase-free 1.5-mL tube. For 100 |L, prepare:

a. 50 |L of deionized formamide.

d. 4 |L of digoxigenin (DIG)-labeled riboprobe (see Note 13). Heat to 65°C for 5 min, then cool on ice for 2 min. Next, add:

a. 20 |L of 5X hybridization buffer.

d. 12.5 mL of RNase- and DNase-free water.

13. Carefully pipet 18 pL of hybridization mixture onto each dried cell spot.

14. Cover with clean cover slip (see Note 14) and seal edges completely with rubber cement to prevent drying out of the cell spot.

15. Denature target DNA by placing slides onto a heating block at 98°C for 8 min before cooling on ice for 2 min (see Note 15).

16. Reseal cover slips and hybridize at 47°C overnight.

17. Carefully remove the rubber cement and cover slip (see Note 16).

18. Wash as follows (with gentle shaking):

a. 30 min in 2X SSC at room temperature (1000 mL).

b. 30 min in 0.1X SSC at room temperature (1000 mL).

c. 30 min in washing solution, 47°C (50 mL in Coplin jar).

d. 15 min in 0.1X SSC at room temperature (1000 mL).

19. Wash 5 min in buffer 1 (50 mL in Coplin jar with gentle shaking).

20. Block for 30 min in buffer 2 (50 mL in Coplin jar with gentle shaking).

21. Wash twice for 15 min in buffer 1 (50 mL in Coplin jar with gentle shaking).

22. Wash once for 5 min in buffer 3 (50 mL in Coplin jar with gentle shaking).

23. Place slides into a fresh Coplin jar containing 30 mL developing solution (see Note 17).

24. Incubate in the dark (without shaking) until signal develops (15 min to 6 h; see Note 18).

25. Stop developing reaction by washing five times briefly with double distilled water (see Note 19).

3.3. In Situ Amplification and Detection of Latent CMV RNA in Myeloid Progenitor Cell Suspensions by RT-PCR-ISH

1. Wash cells (see Note 3) three times in PBS (see Note 4).

2. Resuspend cells in 4% paraformaldehyde (see Note 5) and fix for 30 min at room temperature.

3. Wash cells three times in PBS (see Note 6).

4. Resuspend cells in 200 pL of DNase digestion mixture.

5. Incubate overnight at 37°C.

6. Wash cells three times in PBS.

7. Resuspend cells in 95 pL of reverse transcription buffer.

8. To each 95 pL of mix + cells, add either 5 pL of reverse transcriptase (SuperScript II) or, as a negative control, add 5 pL of RNase- and DNase-free water.

10. Wash cells three times in PBS. For the final wash, transfer cells to 0.5-mL PCR tubes, and split cells as appropriate to include a -Taq polymerase control.

Continue with PCR amplification and detection as described from step 4 onward in Subheading 3.2., with the following modifications: (1) Instead of primers IEP3A and IEP3B, use primers IEP2D and IEP1G for the PCR amplification, and (2) the detection of in sita-amplified products uses a different DIG-labeled riboprobe (see Notes 20 and 21).

4. Notes

1. Store stock solutions of NBT and BCIP at -20°C.

2. Primer IEP2D 5'-CAGGATTATCAGGGTCCATCTTTCTCTTGG-3' and IEP1G 5'-ATAGCAGAGCTCGTTTAGTGAACCG-3' are derived from within exon 2 or exon 1, respectively, of the CMV IE1/IE2 region (17).

3. There will be some loss in cell number during, for example, spins and washes. Although this procedure can be conducted with significantly less cells, a starting of greater than 105 cells per PCR is recommended.

4. Ensure that washing buffer is magnesium-free PBS because the presence of magnesium may significantly alter the subsequent PCR amplification step.

5. Only use freshly prepared 4% paraformaldehyde solution (room temperature) made in PBS. Dissolving paraformaldehyde requires raising the pH with 10 N sodium hydroxide. After the paraformaldehyde has completely dissolved, pH can be returned to neutral with concentrated HCl.

6. All washes of cells are performed in 0.5-mL tubes (or similar), with cells being pelleted using a swing-out rotor. If a 0.5-mL tube rotor is not available, the 0.5-mL tubes can be placed into a larger tube, for instance, 15- or 50-mL conical centrifuge tubes, and centrifuged using a larger rotor. If PCR is to be performed using a 96-well format PCR block, then the same steps can be conducted in smaller PCR tubes. It is worth doing trial centrifugations to ensure that thin walled PCR tubes will not fracture during the washing steps.

7. Primers IEP3A 5'-GTGACCAAGGCCACGACGTT-3' and IEP3B 5'-TCTGCCA GGACATCTTTCTC-3' are derived from within exon 3 of the IE1/IE2 region of CMV (3). Primer design for PCR amplification: The PCR product size for the detection of CMV DNA is 167 bp. Using this method, significantly larger PCR products (e.g., 1 kb) were found to be very difficult to successfully amplify in situ.

8. After 4 min of denaturation at 94°C, pause the machine and add 2 ||L of Taq DNA polymerase. Continue the denaturation for 4 min before thermacyling.

9. After air-drying, slides can be stored in a slide box in at -20°C. The slides can be stored this way until being ready to do the ISH.

10. A staining dish positioned in ice works well for this step.

11. Prepare immediately before use in water with vigorous mixing.

12. Air-dry in a dust-free environment. The cell spots are now ready for the addition of the hybridization mixture. The hybridization mixture can be prepared while the cell spots are drying.

13. Detection of in situ-amplified PCR products was performed using a nonisotopic DIG-labeled riboprobe generated from a genomic clone, p0N2810, which consists of a 2.2-kb A/wNI restriction fragment from the IE1/IE2 region of human CMV strain AD169 cloned into the SmaI site of pBluescript KS (+/-).

14. Cover slips often are contaminated with oils from the manufacturing process and are therefore best thoroughly cleaned before use with an acid wash or detergent solution.

15. After heating to 98°C for 8 min, immediately transfer the slides to a precooled surface for 2 min. A thin metal plate placed onto a bed of ice works well for this cooling step.

16. When removing the cover slip, it is important to not disrupt the cells on the slide or allow any of the hybridization solution to dry onto the slide. Thus, after removing the rubber cement from around the cover slip, place the slide with cover slip still in place into a slide rack and immerse in the first wash buffer. The cover slip can then be gently removed with a pair of fine forceps.

17. Developing solution should be made fresh, immediately before use.

18. Signal development can be checked periodically by removing from developing solution and very briefly examining the slides under a light microscope (with the lamp intensity lowered, as the developing solution is somewhat light-sensitive).

19. After stopping the reaction, slides should be left in double distilled water or cover slipped with a water-based mounting medium. Do not use alcohol or other solvent-based mounting media because they will remove positive staining.

20. In site-amplified RT-PCR products were detected by ISH using a DIG-labeled riboprobe derived from pON2501, which contains a 1.1- kb EcoRVISpeI complementary DNA fragment from the IE1IIE2 region of CMV strain AD169 cloned into the ClaIISpeI site of pBluescript KS (+I-).

21. Primer design for reverse transcription: The design of the RT primers was such that the RT step was across a large spliced region (intron). This was done to bias the PCR reaction in favor of amplification of the complementary DNA rather than a much larger genomic template, in addition to the DNase digestion.

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