Viruses compared to cells

Some ambisense viruses package copy genomes that can be used as templates for transcription, such that the full complement of viral genes can be transcribed soon after infection. It should be noted that packaged copy genomes are not mRNAs and are not translated. Reoviruses also have segmented genomes, packaging 11—12 segments of dsRNA. Reoviruses are nonenveloped and particles consist of two or three concentric icosahedral capsid layers.

A unique feature of the reovirus replication cycle is that the genome segments are transcribed from within the capsid. The genomes of RNA viruses have some common general features. Obviously there are one or more open reading frames that encode the viral proteins.

But there are also regions of RNA that do not code for protein. These non-coding regions NCRs or untranslated regions UTRs are often highly conserved within a virus family, indicating that they have important functions. NCRs may have specific, critical nucleotide sequences but in some cases they are regions of the genome that fold into conserved structures, and structure may be more critical than a specific sequence.

Of course a source of RdRp must be supplied. RdRp may be encoded in the minigenome or may be supplied in trans by using a cell line stably expressing the viral RdRp, for example. The sequences required to direct RNA replication are often fairly simple and can be linked to virtually any RNA sequence to drive its replication.

These promoter sequences can be rather short but provide a means to direct the RdRp to internal sites on the genome. There may also be specific RNA sequences that signal polyadenylation. There are a variety of different strategies that RNA viruses use to regulate transcription and genome replication, but all involve RNA sequences found in the genome.

The RNA genomes of some viruses are highly structured and extensively base paired. The IRES serves as a platform for ribosome assembly. Promoters can be quite long and complex and promoter regions themselves are not transcribed.

It is particularly important, in the case of genome synthesis, that genetic information not be lost or modified; however, mRNAs are often capped and polyadenylated. Are the methods for priming viral mRNA synthesis the same or different from the methods of priming genome replication?

The RNA viruses seem to have experimented widely. For example, the picornaviruses use poly A tracts encoded in the genome.

Among the negative-strand RNA viruses, those in the order Mononegavirales use a stuttering mechanism to synthesize long poly A tracts from short poly U tracts Fig. A strategy to regulate mRNA synthesis. This figure shows the organization of a paramyxovirus genome paramyxoviruses are members of the order Mononegavirales ; negative-strand RNA viruses with unsegmented genomes.

Each protein-coding region is flanked by regulatory sequences that control capping and polyadenylation. The order of the genes on the genome regulates the relative quantities of mRNAs synthesized. Because RdRp does often dissociate from the genome during transcription, the downstream genes are produced in lower quantities.

Even with fairly simple genomes, RNA viruses must, and do, regulate the amounts of genome, copy genome, and mRNAs that are synthesized during an infection. It is much more efficient to synthesize many genomes from each copy genome. Internal promoters for mRNA synthesis can vary in sequence, controlling the relative affinity of the transcription complex for each mRNA.

An important feature of RNA viruses is that many exist in nature as quasispecies. The term quasispecies is used to describe a group of closely related, but nonidentical genomes Fig. A Positive-strand RNA viruses exist as quasispecies, complex mixtures of related genomes. The mixture is more fit than any individual genome; fitness is maintained by generation of new variants in response to selective pressures.

B Potential for safer vaccines. If the fidelity of RdRp is increased the population remains more homogeneous. Therefore an attenuated virus with a high-fidelity RdRp is more likely to remain attenuated. Poliovirus PV is a good example of a virus that forms a quasispecies.

If one examines genome sequences from a mouse experimentally infected with PV serotype 1, we find that the genomes are not identical, although they are all clearly related to one another. To the surprise of many virologists, it turns out that the population quasispecies may be more fit than any individual genome. Or put another way, we cannot find any single genome in the population that replicates better than the group as a whole and in fact, most individual genomes replicate more poorly than the group.

Why this occurs is not always clear, but an animal is a very complex ecosystem. Different members of the quasispecies may be better adapted to different niches in the animal. How does a quasispecies form? But as the cloned virus replicates, mutations accumulate generating a quasispecies. Measurable levels of mutation occur because the fidelity of PV RdRp is low.

Briefly, for the stacking gel, 0. Overlay agarose: 0. 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. 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.

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. A Flow cytometry analysis of mock and avian coronavirus-infected cells at 0 and 16 h post infection. B Detection of avian coronavirus protein by indirect immunofluorescence in infected cells. Vero cells were infected with coronavirus for 0 h or 16 h, fixed, and analyzed by indirect immunoflourescence using appropriate antibodies.

The data indicate that infected cells accumulate in S-phase compared with mock infected cells. To harvest cells for flow cytometry, remove the growth medium and wash the monolayer twice with 3 mLPBS. To remove the remaining cells, scrape using a cell scraper Sarstedt.

The supernatant is removed, and the cells are processed for flow cytometry to detect either incorporated BrdU or cell cycle marker proteins. Fixed and stained cells are analyzed by flow cytometry with a fluorescence-activated cell sorter FACScan; Becton Dickinson; or equivalent.

Ten thousand events per sample should be collected, stored, and analyzed using CellQuest software Becton Dickinson. 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.

Cells can be harvested by trypsinization and rinsed with PBS as described. The supernatant can be discarded. 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.

Overall yields of virus can be determined by plaque assay. 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.

The following nonisotopic Northern blot protocol has been used in our laboratory for the detection and analysis of coronavirus-derived RNA species. An example of what purified RNA looks like by nondenaturing agarose electrophoresis is shown in Fig. When handling RNA, particular care should be taken to avoid contamination see Note 4.

The size of the RNA species is indicated in kb. 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. The upward capillary transfer method is set up as shown in Fig. A 3MM paper wick is cut so that it allows transfer of buffer from the reservoir.

Diagram showing the arrangement of apparatus for upward capillary transfer for Northern blot. SSC, standard saline citrate. 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. 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 h for transfer to occur.

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. 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.

Labeled RNA species present on the membrane are then detected following h of exposure to photographic film see Note 9. 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.

An appropriate dilution series of the virus sample is performed, typically in fold dilution steps, for example, addition of uL of neat virus supernatant to uL of Vero media to make a 10 -1 dilution, and so on. Aspirate media from Vero cells grown to confluency in 6-well plate dishes. Duplicate infections per dilution are typically performed with uL of innoculum per well of virus. 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 Plaques can be visualized, on a light box, as clear zones against a red background.

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 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 G1 arrest; a number of viruses target this protein to usurp its functions.

Vero cells are either treated or not treated with mM etoposide Sigma; as a positive control and left for 48 h. Cells are analyzed for the distribution of caspase 8, following a protocol published in ref. Cover slips are rinsed twice with PBS and permeabilized with 1 mL 0. The blocking solution is removed, and rabbit polyclonal anticaspase 8 P cat. Excess antibody is removed by washing the cover slips in 2 mL PBS three times for 10 min each prior to mounting the cover slips on microscope slides using mounting media containing DAPI Vector.

Mounted coverslips can be analyzed using an Axiovision system Carl Zeiss Jena. Pictures are captured with an Axiocam camera and processed using the Axiovision 3. Figure 4 provides an example of the distribution of caspase 8 in wild-type cells, cells expressing the avian coronavirus nucleoprotein which we know has cell cycle effects and redistributes nucleolar proteins , and control cells.

As can be seen from the figure, caspase 8 is redistributed to the nucleolus in cells expressing a viral protein IBVNHis from an expression plasmid. Localization of caspase 8 in transfected or etoposide-treated Vero cells or cells expressing the avian coronavirus nucleoprotein.

Vero cells were transfected with pTriExIB VNHis a construct that expresses the nucleoprotein under the control of a PolII promoter , treated with mM etoposide, or left untreated and analyzed by indirect immunofluorescence for the localization of caspse 8 using a polyclonal anticaspase 8 P 20 antibody Santa Cruz Biotechnology.

Transfected cells were analyzed 24 h post transfection, whereas etoposide-treated cells were analyzed 48 h post treatment. In nontreated cells left row caspase 8 can be detected in both the nucleus and the cytoplasm, in cells transfected with IB VN His middle row , caspase 8 is localised in the cyto-and nucleoplasm with a prominent signal in the perinuclear region and the nucleolus.

In Etoposide treated cells right row caspse 8 is almost exclusively localised in the nucleus and the perinuclear region. For flow cytometry of caspase 8 expression, Vero cells either treated with m M Etoposide for 48 h for transfected for 24 h either with pTriEx 1.

The supernatant containing detached cells is transferred to canonical centrifuge tubes instead of being discarded. Cells are then resuspended in U.

Cells are permeabilized by resuspending them in 1 mL of PBS with 0. The presence of caspase 8 can be detected by using rabbit polyclonal antibody against caspase 8 P ; Santa Cruz Biotechnology and goat antirabbit FITC-conjugated antibody ; Sigma. 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. In this example we are using Vero and HeLa cells.

Cover slips should be air-dried and washed once with 2 mL PBS prior to addition of 3. To remove the fix, cells are permeabilized with 0. Proteins can be visualized as in Subheading 3. As can be seen in Fig. Thus the antibodies can be used to detect proteins in nonspecies-specific cell lines.

Shown are representative dot plots or mock-transfected cells expressing N protein, control, and IBV Beaudette-infected cell populations. Averages of three experiments for infected cells are shown in the accompanying charts. 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.

In our case we have used Vero cells as an example, as these support avian coronavirus infection. The following protocols are based on original procedures used by Anderson et al. 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.

Prior to harvesting cultured cells for the isolation of nucleoli, rinse each flask three times with prewarmed PBS. In the former case, swirl the flask and return to the incubator for min until most cells have detached.

Check that cells are detached using phase contrast microscopy. Once most of the cells are detached, add a volume of culture media at incubator temperature and pipet up and down so that a single-cell suspension is generated. This suspension can now be placed into Falcon tubes for further steps. To wash the cell suspension, spin samples using a swingout rotor at g relative centrifugal force RCF; e.

Transfer the cell suspension to a precooled tissue homogenizer and homogenize 10 times using a tight-fitting pestle 0. 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. Resuspend the pellet with a volume S1 solution by pipeting up and down. A viral infection will therefore be systemic , spreading throughout the body.

Pathogenic bacteria have a more varied operation and will often infect when the right opportunity arises, so called opportunistic infection. The infection caused by pathogenic bacteria is usually confined to a part of the body, described as a localized infection. These infections may be caused by the bacteria themselves or by toxins endotoxins they produce. Examples of bacterial disease include pneumonia , tuberculosis , tetanus , and food poisoning.

Viruses can infect bacteria. Bacteria are not immune to viral hijackers which are known as bacteriophages —viruses that infect bacteria. Image by Venngage Infographic Maker. Sardoodledom Scripps Bee clip goes viral. Subscribe to America's largest dictionary and get thousands more definitions and advanced search—ad free! Log in Sign Up. Usage Notes 'Virus' vs. What to Know While both can cause disease, viruses are not living organisms, whereas bacteria are.

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