key: cord-272579-aenuyht0
authors: Emmett, Stevan R.; Dove, Brian; Mahoney, Laura; Wurm, Torsten; Hiscox, Julian A.
title: The Cell Cycle and Virus Infection
date: 2005
journal: Cell Cycle Control
DOI: 10.1385/1-59259-857-9:197
sha: 
doc_id: 272579
cord_uid: aenuyht0

A number of different viruses interact with the cell cycle in order to subvert host-cell function and increase the efficiency of virus replication; examples can be found from DNA, retro, and RNA viruses. The majority of studies have been conducted on DNA and retroviruses whose primary site of replication is the nucleus, but increasingly a number of researchers are demonstrating that RNA viruses, whose primary site of replication is normally the cytoplasm, also interfere with the cell cycle. Viral interference with the cell cycle can have a myriad of different effects to improve virus infection, for example to promote replication of viral DNA genomes, or to delay the cell cycle to allow sufficient time for RNA virus assembly. Although cell cycle control is fairly well characterized in terms of checkpoints and control molecules (e.g., cyclins), in recent years several researchers have demonstrated that the nucleolus is also involved in cell cycle control. The nucleolus and associated subnuclear structures can sequester cell cycle regulatory complexes, and nucleolar proteins also have a direct and indirect effect on the cycling cell. Viruses also interact with the nucleolus. In order to study the interactions between a virus and the cell cycle and vice versa we have developed and adapted a number of different approaches and strategies. These include determinations of virus yield and measurements of virus replication to flow cytometry and confocal analysis of the host cell. Increasingly we have found that proteomic approaches allow the rapid analysis of a whole plethora of cell cycle proteins that may be affected by virus infection.

A common strategy of viruses is the creation inside the cell of an environment favorable for efficient replication of their genomes; one way of achieving this is by altering the cell cycle. The function of the cell cycle itself comprises a highly con-served and sequential series of checkpoints and phases ensuring that conditions are suitable for the proper function of that cell and for DNA replication and cytokinesis. There are three categories of viruses, depending on their genome and replication strategy: DNA, retro, and RNA viruses. DNA viruses employ a number of mechanisms to modify and interfere with the cell cycle regulatory machinery. In some cases viruses are adapted to multiply in resting cells, whereas in others they induce proliferation of arrested cells or just wait until the infected cell replicates.

Two main strategies for DNA viruses interfering with host cell cycle control can be distinguished. One has generally evolved in viruses with large genomes with the potential to encode many proteins, including those required for viral DNA replication. A typical example of this strategy is found in the herpesviruses to block cell cycle progression, preventing entry into S-phase (1). The other strategy to impinge on cell cycle control is more typical of DNA viruses with small genomes. Here, virus-encoded proteins, which are not homologs of any known cellular protein, interfere directly with the normal function of cell cycle regulatory components to subvert their activity. Typical examples are the viral oncoproteins that sequester the retinoblastoma (RB) tumor suppressor protein as a first step in inducing S-phase entry by activating the expression of E2F-regulated genes; for example, adenoviruses (2). Disruption of the cell cycle to favor virus replication is not confined to DNA viruses. Retroviruses also disrupt the cell cycle.

Cells infected with human immunodeficiency virus (HIV) do not proliferate but accumulate in the G 2 -phase of the cell cycle, resulting in an increase in virus production (3). The viral protein responsible for this has been identified as Vpr (4). Interestingly, Henklein et al. (5) demonstrated that extracellular added Vpr induced G 2 -phase arrest, and the authors suggested that free Vpr in the serum of HIV-infected patients may preprogram cells in order to facilitate replication of HIV in infected individuals. Consistent with the perturbation of the cell cycle observed in tissue culture-infected cells, lymphocytes from HIV-infected individuals show high levels of cyclin B and also nucleolar proteins (whose expression is linked to the cell cycle) (6) .

Altering the host cell cycle by RNA viruses has not been described extensively in the literature, and the mechanisms of action are generally not well characterized. For the negative-strand RNA viruses, there are several examples of cell cycle control. For instance, measles virus infection results in a G 0 block (7), and the paramyxovirus simian virus V protein prolongs the cell cycle by delaying cells in G 1 and G 2 (8). In the case of positive-strand RNA viruses, the avian coronavirus infectious bronchitis virus (IBV) delays cell growth by inhibiting cytokinesis and also allows cells to accumulate in S/G 2 (9, 10) . Several examples of different picornaviruses interacting with the cell cycle have been described. More recently Feuer et al. (11) have shown that cells arrested in G 1 or G 1 /S produced higher levels of infectious coxsackievirus and viral polyproteins compared with cells in the G 0 phase, or cells blocked at the G 2 /M boundary. Feuer et al. (11) suggested that persistence of coxsackie B3 virus (CVB3) in vivo might be dependent on infection of G 0 cells, which do not support replication. If such cells were to reenter the cell cycle, then the virus may reactivate and trigger chronic viral or immune-mediated pathology in the host. Such findings suggest that locally delivered cell cycle inhibitors could form part of an antiviral therapy, similar to the example of interleukin-2 used to correct cell cycle aberrations in HIV-infected individuals (12).

Viruses also target subnuclear structures involved in cell cycle regulation as part of a strategy to subvert host cell functions such as the cell cycle. For example, many DNA, retro, and RNA viruses target the nucleolus (13,14) . The nucleolus and associated proteins are also implicated in (and regulated by) the cell cycle (15). Cajal bodies associated with nucleoli can sequester cyclin-dependent kinase 2 (CDK2) and cyclin E in a cell cycle-dependent manner (16). The concentrations of many nucleolar proteins such as nucleolin (17) are dependent on the cell cycle (18, 19) . Nucleolin itself can also act as a cell cycle regulator (20) .

When studying viruses and the cell cycle, one must consider the interrelationship between the two. The cell cycle will have an effect on virus replication, and virus replication will concomitantly affect the cell cycle. Below we describe experiments that can be used to investigate whether a particular virus interacts with the cell cycle or vice versa. These range from traditional approaches toward measuring the cell cycle and virus production to the use of confocal microscopy to investigate the redistribution of cell cycle factors in virus-infected cells to a proteomic analysis of the nucleolus, which has recently been identified as having roles in cell cycle regulation (21,22). 

There are two options for investigating the cell cycle stage and affect on virus replication. The first is to synchronize cells using serum starvation, release the cells, and determine the cell cycle profile by labeling the cells with bromodeoxyuridine (BrdU) and propidium iodide and analyzing on a flow cytometer. Cells can then be infected at different stages of the cell cycle; depending on the length of the virus life cycle virus replication can occur within a particular cell cycle stage. The second alternative is to use cell cycle inhibitors to block a particular stage of the cell cycle. This has the advantage that cells can be inhibited for prolonged lengths of time, perhaps sufficient to cover the infections cycle; it also has several disadvantages, including the fact that not all cells will arrest at the same stage 100% of the time, and cell cycle inhibitors may affect viral processes themselves.

Simple experiments can be performed to investigate whether a virus has an effect on cellular proliferation and/or the cell cycle. For example, a Coulter counter can be used to measure cellular proliferation in infected compared with noninfected cells (10) and the cell cycle profiles of infected cells compared with noninfected using flow cytometry (Fig. 1) . Infection protocols vary from virus to virus, and these can be found by consulting specific literature. Below we detail our protocols for determining the cell cycle stage. To detach the cells, add 2 mL of PBS/EDTA/trypsin and incubate at 37°C for 5 min or until cells detach (see Note 1). 3. To remove the remaining cells, scrape using a cell scraper (Sarstedt). To inactive the trypsin, transfer by pipeting into a canonical tube filled with 8 mL 10% DMEM. 4. The suspension is then centrifuged at 250g for 10 min at 4°C, the supernatant is removed, and the cell pellet is resuspended in 2 mL ice cold PBS prior spinning at 250g for 10 min/ 4°C. 5. The supernatant is removed, and the cells are processed for flow cytometry to detect either incorporated BrdU or cell cycle marker proteins.

1. Fixed and stained cells are analyzed by flow cytometry with a fluorescence-activated cell sorter (FACScan; Becton Dickinson; or equivalent). 2. Ten thousand events per sample should be collected, stored, and analyzed using CellQuest software (Becton Dickinson).

1. To determine the proportion of cells undergoing DNA replication, cells are pulsed by addition of 10 mM BrdU, an analog of thymidine, for 30 min prior to harvesting. 2. Cells can be harvested by trypsinization and rinsed with PBS as described. After centrifugation at 250g for 10 min at 4°C, the pellet is suspended in 1 mL of precooled (-20°C) 70% ethanol (see Note 2). 3. To remove the fix, cells are centrifuged at 300g for 10 min at 4°C and the ethanol removed. 4. To denature the DNA, 500 μL of 0.1 M HCl is added and incubated at 37°C for 15 min. 5. The reaction is then stopped with the addition of 3 mL PBS and centrifugation at 300g for 10 min/4°C. 

There are several stages to the virus life cycle, and these include attachment to the host cell, entry, uncoating of the genome, transcription and translation of viral mRNAs, replication of the genome, packaging of new genomes, virus assembly, and release of new virus particles. Several assays can be used to measure these different stages of virus infection; however, a simple analysis measuring replication and total virus production will provide information as to whether a cell cycle stage affects virus infection. One of the simplest ways to measure virus replication is to use Southern (in the case of DNA viruses) and Northern (in the case of RNA viruses) blots. Overall yields of virus can be determined by plaque assay. Using an RNA virus as an example, we detail two protocols that can be used to measure RNA replication (mRNA production) and amount of virus. These can be readily adapted to study other viruses. We routinely work with coronaviruses, which are positive-strand RNA viruses, and principally cause respiratory infection (e.g., severe acute respiratory syndrome.

The following nonisotopic Northern blot protocol has been used in our laboratory for the detection and analysis of coronavirus-derived RNA species. RNA is routinely obtained by extraction of cytoplasmic RNA from coronavirus-infected cells using the Qiagen™ mini-prep RNA purification kit. For longer RNA species, or for the preparation of RNA that is free from DNA or from tissues and organs, we use the Promega RNAgents ® total RNA isolation system coupled with multiple freeze-thaws and homogenization, and routinely include a DNase (RQ1 DNase, Promega) treatment step (see Note 3). (An example of what purified RNA looks like by nondenaturing agarose electrophoresis is shown in Fig. 2A.) In this latter case, all purification steps are conducted at 4°C (i.e., in a cold room). When handling RNA, particular care should be taken to avoid contamination (see Note 4).

1. Excess agarose gel not containing any potential RNA species is trimmed from the gel and nylon membrane, and approx 10 pieces of 3MM blotting paper are cut to the same size as the gel (see Note 7). 2. The upward capillary transfer method is set up as shown in Fig. 3 . A 3MM paper wick is cut so that it allows transfer of buffer from the reservoir. 3. The 3MM paper wick is presoaked in 20X SSC buffer, and the nylon membrane and three sheets of 3MM paper are presoaked in 2X SSC prior to capillary transfer assembly. RNA extracted from Vero cells using the Promega RNAgents ® total RNA isolation system. This is a nondenaturing 1% agarose gel, and the RNA is visualized by staining with ethidium bromide. In good RNA preparations, the ratio of 28s rRNA to 18s rRNA should be approx 2:1. (B) Northern blot analysis of total cytoplasmic RNA extracted from avian coronavirus (IBV B-US)-infected chick kidney (CK) cells. RNA was extracted from IBV B-US-infected CK cells, separated by electrophoresis on a 1% denaturing formaldehyde agarose gel, and transferred to nylon membrane. IBV-derived RNAs were detected by hybridization with an IBV 3' untranslated region genomic probe capable of detecting IBV genomic RNA and IBV mRNAs. The black arrowheads indicate the positions and sizes of the IBV B-US subgenomic mRNAs. The size of the RNA species is indicated in kb. 4 . The agarose gel is oriented so that the wells are facing downward to minimize the distance RNA has to migrate through the agarose gel to the nylon membrane. 5. Sufficient dry paper towels must be used draw enough transfer buffer through the gel and membrane to allow efficient transfer to occur. The transfer apparatus is weighed down with a 1-kg weight and left overnight (8-16 h) for transfer to occur. 6. Once transfer is complete, the nylon membrane is washed briefly in 2X SSC buffer, and the RNA is UV-crosslinked (254 nm UV light for 25-50 s) to the nylon membrane (see Note 8).

In recent years nonradioactive detection of nucleic acid species has been as effective as with radioactive methods. The nonradioactive methodologies have a number of advantages, mainly associated with health and safety issues. We routinely use DNA probes nonisotopically labeled using the Ambion BrightStar Psoralen-Biotin kit. The label is composed of a planer, tricyclic psoralen compound covalently attached to biotin. Psoralen intercalates between nucleic acids and covalently binds during irradiation by long-wave UV light to create biotin-labeled DNA.

1. For each labeling reaction 10 μL of purified DNA template (0.5 ng/μL-0.5 μg/μL) is used, producing five 20 μL DNA-labeled probes. Probes are stored at -80°C. 2. Crosslinked membrane is prehybridized prior to probing in 10 mL pre-hybridization/hybridization solution for 30 min at 42°C within a hybridization tube in a roller oven. 3. Following prehybridization, a 20 μL prepared probe aliquot is diluted to 100 μL with nuclease-free water, incubated at 100°C for 10 min, and then added to the hybridization tube. 4. Membranes are incubated at 37°C overnight (approx 8-16 h). Following hybridization, membranes are washed twice with 20 mL of 2X SSC/1% SDS low-stringency wash solution at 42°C for 5 min and then twice with 20 mL of 0.2X SSC/0.1% SDS high-stringency wash buffer at 42°C for 5 min. 

Hybridized psoralen-biotin-labeled DNA probe is detected using the Ambion chemiluminescent non-isotopic BrightStar BioDetect kit according to manufacturer's instructions. Hybridized biotinylated DNA probe is bound by a streptavidin/alkaline phosphatase conjugate; the blot is then incubated with detection reagent, resulting in chemiluminescence of labeled probe. Labeled RNA species present on the membrane are then detected following 2-4 h of exposure to photographic film (see Note 9). An example of a Northern blot of viral RNA is shown in Fig. 2B .

The following protocol has been successfully used to quantitatively determine the number of infectious avian infectious bronchitis coronavirus particles within a sample by plaque assay using Vero cells within our laboratory. This method can be adapted for calculating the titer of a variety of viruses that induce cytopathic effect in cell culture by adapting the cell type, media, and environmental conditions according to the particular virus. For example, the number of baculovirus infectious particles can be calculated by plaque assay using Grace's insect medium supplemented with FBS on Sf9 cells.

1. An appropriate dilution series of the virus sample is performed, typically in 10-fold dilution steps, for example, addition of 150 μL of neat virus supernatant to 1350 μL of Vero media to make a 10 -1 dilution, and so on. 2. Aspirate media from Vero cells grown to confluency in 6-well plate dishes. Duplicate infections per dilution are typically performed with 500 μL of innoculum per well of virus. 3. Plates are incubated for 1 h at 37°C within a 5% CO 2 incubator (see Note 10). 4. During the 1-h incubation, preheat the Vero media to 37°C. 5. Melt the 2% low melting point agarose solution within a microwave or boiling water bath until the agarose is completely in solution, and then equilibrate to 42°C. 6. At 1 h post infection, aspirate media from cells, and wash twice with PBS. 7. Mix an equal volume of equilibrated media and agarose solution and overlay the cells with 2 mL of solution by carefully pipeting the solution down the side of the well (see Note 11). 8. Allow the agarose overlay to solidify fully, and then incubate the plates for 3 d at 37°C

within a 5% CO 2 incubator. 9. At 3 d post infection, prepare media/agarose solution and add 1% neutral red stock solution to a final concentration of 0.01% neutral red. 10. Overlay each well with 2 mL of the media/agarose/neutral red solution, and allow to set fully before incubating the plates for 1 d at 37°C within a 5% CO 2 incubator. 11. Plaques can be visualized, on a light box, as clear zones against a red background. The virus titer can then be calculated at a particular dilution as the number of plaque forming units (PFU)/mL (see Note 12):

Titer (PFU/mL) = average number of plaques × 1/vol of innoculum (mL) × 1/dilution.

For example: an average of 25 plaques from 500 μl of innoculum per infection at a dilution of 10 -6 .

Titer (PFU/mL) = 25 × 1/0.5 × 1/10 -6 = Titer of 5 × 10 7 PFU/mL

Viral effects on the cell cycle can often be attributed to interference with cell cycle factors, such as the cyclins. Here we detail our approaches to investigating the effect of virus on what some might consider nonconventional cell cycle factors, caspase 8 and proliferating cell nuclear antigen (PCNA). Viral effects on these factors (as with any factor) should be studied both at the level of their cellular localization, which in turn can affect function, and also for analysis of expression levels in the cell. To study localization we routinely use confocal microscopy, and to study expression levels, we use flow cytometry. Microscopy has the added advantage that it can be used to check that antibodies recognize proteins of interest in both species-specific and -nonspecific cells. Both monoclonal and polyclonal antibodies to various mammalian proteins can be crossreactive depending on the degree of conservation between the various proteins. For example, a monoclonal antibody to fibrillarin (a nucleolar protein) can be used to detect fibrillarin via immunofluorescence in human, monkey, and avian cells (9).

Recent work has identified and highlighted the role of caspases in cell proliferation (23). Caspase 8 has been postulated to activate downstream nuclear caspase 3, which in turn cleave various negative regulators of the cell cycle, such as p21 Cip1/Waf1 or p27 Kip (24), thereby leading to activation of CDK2. Activation of CDK2 in turn induces its dissociation from cyclin E or cyclin A, thereby inactivating cyclin E function by degrading cyclin E by the proteasome pathway and subsequent G 1 arrest; a number of viruses target this protein to usurp its functions.

Cells are grown on cover slips, normally in 6-well plates. 

Proliferating cell nuclear antigen (PCNA) expression is associated with S-phase, and its localization is restricted to sites of DNA replication, as shown by immunofluorescence analysis. During DNA replication, PCNA functions as an auxiliary protein for DNA polymerase , and its presence is necessary for synthesis of the leading strand, although the precise function has not been clarified. 2. Cover slips should be air-dried and washed once with 2 mL PBS prior to addition of 3.5% paraformaldehyde in PBS for 30 min at 4°C. 3. To remove the fix, cells are permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 2 min at RT. 4. After extensively washing four times, each time for 10 min, with PBS, the cover slips should be covered with 500 μL of mouse monoclonal anti-PCNA (PC 10) antibody (1:100 in PBS) and incubated for 1 h at 37°C in a humidified atmosphere. 5. Cover slips are then washed three times in 2 mL PBS and stained with 500 μL FITCconjugated secondary goat antimouse (Sigma) antibody (1:100 in PBS). 6. After 1 h of incubation at 37°C, the cover slips are washed three times with 2 mL PBS and mounted using mounting medium containing DAPI (Vectashield, Vector). (Proteins can be visualized as in Subheading 3.3.1.1., step 9 ).

As can be seen in Fig. 5 antibody to human PCNA can detect this protein in both human (HeLa) and Vero (a monkey cell line) cells. Thus the antibodies can be used to detect proteins in nonspecies-specific cell lines. HeLa and Vero cells were grown in 10% DMEM medium on glass cover slips and labeled for PCNA using a mouse monoclonal anti-PCNA antibody (Santa Cruz Biotechnology) as primary antibody and FITC-conjugated goat anti PCNA antibody (Sigma) as secondary antibody. DAPIcontaining mounting medium (Vector) was used to counterstain DNA. In both HeLa and Vero cells, PCNA was detected exclusively in the nucleoplasm..

As can be seen in Fig. 6 the number of cells expressing PCNA in avian coronavirusinfected cells or cells expressing the viral nucleoprotein (when expressed from an expression plasmid) is less than when compared to mock treated cells.

To detect the number of cells expressing PCNA, flow cytometry can be used. Cells were harvested as described above prior to flow cytometry analysis. The protocol used followed essentially a protocol published by ref. 26 with slight modifications. In our case we have used Vero cells as an example, as these support avian coronavirus infection. As can be seen in Fig. 5 the number of cells expressing PCNA in avian coronavirusinfected cells or cells expressing the viral nucleoprotein (when expressed from an expression plasmid) is less than when compared with mock treated cells.

The following protocols are based on original procedures used by Anderson et al. (27) for the isolation of nucleoli from cultured cell lines. The methods presented here have been successfully used in our laboratory for isolation of nucleoli for subsequent proteomic analysis by 2D SDS-PAGE. This latter method can be used, in conjunction with mass spectrometry, N-terminal protein sequencing, or Western blotting, to identify proteins isolated from the nucleoli. As discussed in the Introduction, the nucleolus is targeted by many different types of virus (13, 14) , and such interactions may cause perturbations to the distribution of cell cycle factors (e.g., Fig. 4 ) and the cell cycle. 5. To wash the cell suspension, spin samples using a swingout rotor at 220g relative centrifugal force (RCF; e.g., rotor A-4-62 Eppendorf, 220g RCF, 1046 rpm), remove supernatant so that a cell pellet remains, and add a volume of 4°C PBS. Carry out this process a further two times before resuspending cells in HEPES (see Subheading 2.3., item 4 and see Note 13). 6. Transfer the cell suspension to a precooled tissue homogenizer and homogenize 10 times using a tight-fitting pestle (0.0010-0.0030-inch clearance), while keeping the homogenizer on ice. The number of strokes needed depends on the cell type used, so it is necessary to examine the homogenized cells with a phase contrast microscope after every 10 strokes. Stop when >90% of the cells have burst, leaving intact nuclei. 7. To obtain a pellet containing enriched nuclei, centrifuge the homogenized cells again at 220g for 5 min at 4°C. 8. Resuspend the pellet with a volume S1 solution by pipeting up and down. In another tube containing a volume of S2, layer onto the top the resuspended pellet; ensure that the two layers remain cleanly separated. 9. Centrifuge this cushion at 1430g for 5 min at 4°C to obtain a purer nuclear pellet. Following this spin, discard the supernatant, and resuspend the pellet in a volume of S2 solution by pipeting up and down. 10. Appropriate sonication on ice is a crucial stage in the preparation of nucleoli (see Note 14). 11. To prepare a nucleolar concentrated pellet, layer the sonicated material over a volume of S3 solution and centrifuge at 3000 g for 10 min at 4°C. 12. Further purification of nucleoli can be carried out by re-suspending the pellet with S2 solution, and centrifuging at 1430g for 5 min at 4°C. The resulting pellet contains highly purified nucleoli, which can be examined by microscopy and, if required, stored at -80°C.

The use of 2D PAGE has come into widespread usage since the publication of methods combining isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension. Three separate papers by O'Farrell and others have demonstrated that it was possible to combine IEF with gradient SDS-PAGE gels to separate and reveal proteins better in a gel, thus improving the resolution of 1D SDS-PAGE. Two-dimensional SDS-PAGE is particularly useful for the separation of extremely complex protein mixtures.

The following protocols are based on equipment and materials available from Bio-Rad.

1. Make up prepared nucleoli to 2.5-3 mg total protein in sample buffer. 2. Remove the desired number of pH 4.0-7.0 ReadyStrip IPG strips (Bio-Rad, UK) from the -20°C freezer, and set them aside to defrost. It is good practice to run two sets of strips per sample, one to be stained following the IEF phase, and the other to be used for the 2D and subsequent analysis. 3. Using a suitable tray, place sufficient sample volume into each well so that each IPG strip is in contact with the solution through its entire length. Lay the IPG strip gel side down into the sample buffer. Ensure that all samples are evenly in contact with the strip since it will not be absorbed through the plastic backing. It is recommended that for strips of 11 cm, 185 μL of sample be used per strip (approx 250 μg per strip). When preparing samples, do not place the vials on ice, as the urea will crystallize out of the solution. 4. Leave these strips for 1 hr, then overlay each of the strips with 2-3 mL of mineral oil to prevent evaporation during the strip rehydration process. 5. For rehydration cover the tray with a lid and leave the tray sitting on a level bench overnight (11-16 h) to rehydrate the IPG strips thoroughly with the nucleolar sample; rehydration is crucial to successful 2D.

1. In the IEF tray (Bio-Rad, UK; see manufacturer's instructions), place a paper wick at both ends of the channels covering the wire electrodes. Pipet 8 μL of pure water onto each wick to wet them. 2. Following strip rehydration, remove strips from the incubation tray using forceps, carefully holding the strips at the end where there is no gel, and hold the strip vertically for 7-8 s until the mineral oil has drained. Each strip can then be transferred to the corresponding channel in the focusing tray, gel side down, with the positive end of the strip adjacent to the positive electrode of the tray, in contact with the wetted electrodes. 3. Each of the strips should again be covered with 2-3 mL of fresh mineral oil. 4. The focusing tray can now be placed into the protean IEF cell (Bio-Rad, UK) with the positive side of the tray corresponding to the positive electrode of the cell. 5. Once the cell cover has been closed, the IEF cell can be programmed (see Bio-Rad Protean IEF cell instructions) for a single-or multiple-step focusing protocol. In our laboratory a three-step protocol has proved satisfactory for IEF of nucleolar proteins. Our program follows the basic format of:

Step 1 linear voltage ramp, 250 V for 20 min.

Step 2 linear voltage ramp, 8000 V for 2.5 h.

Step 3 rapid voltage ramp, 20,000 VHrs.

6. When setting up the program, it is satisfactory to use the default IEF cell temperature of 20°C, with a maximum current of 50 μA per strip. This three-step program takes approx 6 h. Once the program has finished, it is important either to place the strips under a 500-V holding voltage or remove, cover with tin foil, and freeze at -80°C or stain for protein.

1. For determining whether proteins have isoelectrically focused correctly, it is possible to transfer IPG strips to a clean, dry piece of blotting filter paper with the gel side up, thoroughly wet a second filter paper of the same size with pure water, and carefully lay the wet filter paper onto the IPG strips. Then "peel" back the top filter paper. This blotting step removes mineral oil on the surface of the IPG. 2. The IPG strips can then be stained for protein using 0.1% (w/v) Coomassie brilliant blue solution (1 h) and then destained with destain buffer until proteins are revealed (1-3 h; see Subheading 2.4.). Alternatively, strips can be stained with Bio-Rad's IEF stain (Bio-Rad, UK).

1. If strips were frozen following the first dimension separation, then remove from the -80°C freezer and allow to defrost thoroughly. It is best to not leave the thawed IPG strips for longer than 15-20 min, as diffusion of the proteins can result in reduced sharpness of the protein spots.

RNase-free tips and prepacked RNase-free tubes. When conducting RNA work, ensure that all liquid dispensers (i.e., Gilson pipets or equivalents) are wiped with 75% ethanol, and wear suitable gloves. Be sure not to touch any part of exposed skin (i.e., your face) with the gloves; this is a bad habit that many laboratory workers have. Many people secrete DNase and RNases. 5. It is advisable to use as thin a gel as possible; the thinner the gel, the faster and more efficient the transfer of RNA to membrane. 6. Running agarose gels for Northern blot analysis at high voltages can result in gel warping owing to heat. It is advisable to run the gel overnight at low voltages to minimize temperatures, and it is advantageous to use a buffer recirculation pump. 7. Touch the nylon membrane as little as possible to prevent nuclease contamination. 8. Once they are crosslinked, membranes can be stored at -20°C for several months. 9. Unlike isotopic 32 P-labeled probes, placing the film cassette containing the membrane exposed to film at -80°C will not increase the signal of weak RNA-labeled species. 10. Every 15 min, gently agitate the plates to ensure that the Vero cells are fully overlaid with innoculum to prevent cell desiccation. 11. Ensure that the agarose is fully equilibrated to 42°C before addition to the media. Addition of agarose solution at temperatures higher than 42°C can damage the cells. 12. Virus titer can vary depending on the cell type used. 13. It is good practice to ensure that cells have not lysed but have become swollen in the buffer conditions by examining with an inverted microscope. Extra care should be taken when working with mammalian cells, as these are particularly prone to lysis at 37°C in hypotonic conditions; therefore preparation on ice is imperative. 14. With most cell preparations, sonication of the nuclear suspension for six 10-s bursts (with 10-s intervals between each burst) has proved sufficient in our hands. In our laboratory we use a Misonix XL 2020 sonicator fitted with a microtip probe set at a power setting of 5. The optimal sonication conditions do, however, depend on the cell types used; oversonication leads to destruction of nucleoli, whereas undersonication leaves the sub- Fig. 7 . Silver-stained 2D gel of purified nucleoli focused in the first dimension using a 3-10 strip (Bio-Rad) and then subsequently subjected to electrophoresis on a 10-20% SDS-PAGE gel. cellular component intact. For best results, examine the suspension by microscopy after each round of sonication; there should be virtually no intact cells, and the nucleoli should be seen as dense, refractive bodies. 

DNase should be removed prior to any cloning by reverse transcriptase (RT)-PCR. A phenol/chloroform extraction followed by ethanol precipitation is suitable for this

at all stages it is highly advantageous to use either nuclease-free or diethyl pyrocarbonae (DEPC)-treated water to make up all solutions. All work areas and equipment should be cleaned with an RNase inhibitor (such as Ambion RNaseZap), and gloves should be worn at all times

Interactions of adenoviral proteins with pRB and p53

Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G 2 by inhibiting the activation of p34 cdc2 -cyclin B

Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents

Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus and induces G 2 cell cycle arrest

Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis

Cell cycle arrest during measles virus infection: a G 0 -like block leads to suppression of retinoblastoma protein expression

The paramyxovirus simian virus 5 V protein slows progression of the cell cycle

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro

Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virusinfected individuals

Brief review: the nucleolus-a gateway to viral infection?

The interaction of animal cytoplasmic RNA viruses with the nucleus to facilitate replication

To be or not to be in the nucleolus

Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies

Amount of the two major Ag-NOR proteins, nucleolin and protein B23 is cell-cycle dependent

Cell cycle redistribution of U3 snRNA and fibrillarin

Effects of antifibrillarin antibodies on building of functional nucleoli at the end of mitosis

Molecular dissection of nucleolin's role in growth and cell proliferation: new insights

The nucleolus: an old factory with unexpected capabilities

Conventional and nonconventional roles of the nucleolus

Apoptosis-independent functions of killer caspases

Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade

Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry

Directed proteomic analysis of the human nucleolus

Protein blotting: principles and applications

with 4 mL of equilibration buffer I for 10 min on a slowly rotating orbital shaker. At the end of the 10-min incubation, discard the used equilibration buffer I in its entirety by carefully decanting the liquid from the tray. To each strip then add 4 mL of equilibration buffer II, and place on an orbital shaker for a further 10 min. 3. During this incubation either prepare SDS-PAGE gels and ensure that the stacking gel is of sufficient size to take the IPG strip, or obtain a suitable number of precast polyacrylamide gels for your samples (see Subheading 2.4.). 4. Once the combs have been removed from the gels, rinse each well briefly with nanopure water using a water bottle. Working quickly, prepare sufficient 1X Tris-HCl/glycine/SDS running buffer to run the number of gels you have decided upon. 5. Once the strips have finished incubating and the gels are prepared, melt sufficient overlay agarose in a microwave to cover the IPG strips once they are inserted into the wells.

1. Ensure that the IPG wells of the gels are free of any liquid by blotting with Whatman 3MM paper. 2. Remove the incubated strip from the incubation tray, and dip each IPG strip into a tube of suitable length to take the entire length containing 1X Tris-HCl/glycine/SDS running buffer. 3. Carefully lay each strip gel side up and onto the back plate of the SDS-PAGE gel above the IPG well, and pipet into the well the liquid overlay agarose. Once the well is full, gently move the IPG strip down until it is in contact with the top of the SDS-PAGE gel (avoid trapping any air bubbles). 4. Allow the agarose to solidify for 5 min before proceeding. 5. Once the agarose has solidified, mount the gel, fill the reservoirs with running buffer, and begin the electrophoresis, run at the appropriate voltage for the gel size used (see manufacturer's instructions; or 150 V for a 14-cm 12% Tris-HCl SDS-PAGE gel).

1. Once the sample front has reach the bottom of the gel, you can proceed to reveal the proteins in the nucleolar sample by using commercially available stains such as Coomassie blue (see Subheading 2.) or silver stain plus (Bio-Rad, UK; carry out staining as recommended by the manufacturer) (Fig. 7) . If you wish to examine individual, known proteins, the gel can simply be blotted onto nitrocellulose or polyvinyl idene difluoride membranes for probing with specific antibodies. For further information on this latter technique of western blotting, see ref. 33.