Viral bioinformatics: Introduction to multiple sequence alignment

This week’s addition to the virology toolbox was written by Chris Upton

Generating multiple sequence alignments (MSA) is one of the most commonly used bioinformatics techniques. The “sequences” to be compared can be DNA (promoters, genes, genomes) or proteins. Note that the length and number of sequences to be aligned has an impact on the methods (algorithms) that can be used; what is suitable for aligning 20 proteins probably won’t work for alignment of 5 poxvirus genomes (200 kb each).

Some useful links:

So you see, there lots of options (did you say: “too many!”?). Further confusion may arise because 1) the same algorithm may be used in many different software programs, and 2) referencing a software package may give no clue to the algorithm used. For many molecular biologists, Clustal is synonymous with sequence alignment. However, newer algorithms such as T-Coffee and MUSCLE are often offered in current software packages, and may be faster and more accurate.

Specialized alignment tools are almost always needed for long, genome sized DNA sequences.

In this set of posts, I’ll provide some information on favorite general MSA tools (that are free) that should be useful to the average molecular virologist. The lists noted above provide a multitude of tools, but many are for specific analyses.

Detecting viral proteins in infected cells or tissues by immunostaining

Many virological techniques are based on the specificity of the antibody-antigen reaction. Examples in our virology toolbox include western blot analysis and ELISA. While very useful, these methods cannot be used to visualize viral proteins in infected cells or tissues. To do that we must turn to immunostaining.

In direct immunostaining (illustrated), an antibody that recognizes a viral antigen is coupled directly to an indicator (a fluorescent dye or an enzyme). Indirect immunostaining is a more sensitive method because a second antibody is coupled to the indicator. The second antibody recognizes a common epitope on the virus-specific antibody. Multiple second antibodies can bind to the first antibody, leading to an increased signal from the indicator compared to direct immunostaining.

To carry out immunostaining, virus-infected cells are fixed to preserve cell morphology or tissue architecture. This step is usually accomplished with acetone, methanol, or paraformadehyde. After incubation of fixed cells with the appropriate antibody, excess antibody is removed by washing, followed by microscopy. Common indicators that are coupled to antibody molecules include fluorescein and rhodamine, which fluoresce on exposure of the cells to ultraviolet light. Filters are placed between the specimen and the eyepiece to remove blue and ultraviolet light; this ensures that the field is dark, except for cells that have bound antibody. These emit green (fluorescein) or red (rhodamine) light.

Antibodies can be coupled to indicators other than fluorescent molecules. Examples are enzymes such as alkaline phosphatase, horseradish peroxidase, and β-galactosidase. These enzymes can convert an added substrate to a colored dye. For example, the bacterial enzyme β-galactosidase converts the chromogenic substrate X-gal to a blue product, which can be visualized by microscopy.

Immunostaining is widely used in the research laboratory to determine subcellular location of proteins in cells. An example is the location of the herpes simplex viral protein VP22 in the nucleus of infected cells. To produce this image, virus-infected cells were stained with an antibody against VP22 and a mouse monoclonal antibody against α-tubulin, a cellular protein. Second antibodies bound to indicator molecules were then added: fluorsecein-conjugated anti-rabbit antibody, and Texas red-conjugated anti-mouse antibody (Texas red is another red fluorescent dye). The stained cells were then photographed with a microscope using ultraviolet light. The results show that VP22 (green) is located in the cell nucleus. Cellular α-tubulin is stained red. Photo courtesy of John Blaho.

Other uses of immunostaining include monitoring the synthesis of viral proteins, determining the effects of mutation on protein production, and investigating the sites of virus replication in animal hosts. Immunostaining of viral antigens in clinical specimens is also used to diagnose viral infections. Direct and indirect immunofluorescence assays with nasal swabs or washes are routine for diagnosis of infections with respiratory syncytial virus, influenza virus, parainfluenza virus, measles virus, and adenovirus.

When cultured cells are examined by this technique it is called immunocytochemistry; when tissues are studied, the procedure is immunohistochemistry. Flow cytometry is yet another way to use immunostaining to study the synthesis of one or more proteins in cells.

Detection of antigens or antibodies by ELISA

A more rapid method than Western blot analysis to detect a specific protein in a cell, tissue, organ, or body fluid is enzyme-linked immunosorbent assay, or ELISA. This method, which does not require fractionation of the sample by gel electrophoresisis, is based on the property of proteins to readily bind to a plastic surface.

To detect viral proteins in serum or clinical samples, a capture antibody, directed against the protein, is linked to a solid support such as a plastic 96 well microtiter plate, or a bead. The clinical specimen is added, and if viral antigens are present, they will be captured by the bound antibody. The bound viral antigen is then detected by using a second antibody linked to an enzyme. A chromogenic molecule – one that is converted by the enzyme to an easily detectible product – is then added. The enzyme amplifies the signal because a single catalytic enzyme molecule can generate many product molecules.

To detect antibodies to viruses, viral protein is linked to the plastic support, and then the clinical specimen is added. If antibodies against the virus are present in the specimen, they will bind to the immobilized antigen. The bound antibodies are then detected by using a second antibody that binds to the first antibody.

ELISA is used in both experimental and diagnostic virology. It is a highly sensitive assay that can detect proteins at the picomolar to nanomolar range (10-12 to 10-9 moles per liter). It is the mainstay for the diagnosis of infections by many different viruses, including HIV-1, HTLV-1, adenovirus, and cytomegalovirus.

Virology toolbox: the western blot

Readers of virology blog often request explanations of specific experimental techniques. Methods such as complement fixation, deep sequencing, ELISA, PCR and many others are frequently mentioned on this blog without discussion. To do so would interrupt the scientific discourse and make for lengthly posts. To remedy this shortcoming, I have added a new tab to the first page of virology blog called Virology Toolbox. This page will be populated with explanations of experimental techniques used for the study of viruses. Today’s technique is the western blot.

Western blot analysis (also known as immunoblotting) is used to detect a specific protein in a cell, tissue, organ, or body fluid. The technique depends on the reaction of an antibody with a protein that is immobilized on a thin membrane (click the figure for a larger version). The sample is solubilized with detergent, and the proteins are then separated by electrophoresis in a polyacrylamide gel. After electrophoresis, the gel is placed next to a thin, synthetic membrane that has a strong affinity for proteins. In the figure, the gel and membrane are placed between sheets of absorbent paper in a blotting tank. This arrangement allows buffer to flow across the gel and through the thin membrane. As a result, the proteins in the gel are transferred to the membrane by capillary action. Transfer of the proteins to the membrane may also be accomplished by an electrical current.

After the transfer step, the membrane is incubated with an antibody to a specific protein. This antibody may be produced in an experimental animal such as a mouse or rabbit, or in cells as a monoclonal antibody. The antibody may be coupled to an enzyme which can then be used to detect the antibody on the membrane. In the example shown, the antibody is coupled to horseradish peroxidase. The membrane is incubated with a substrate that is converted to a luminescent compound after reaction with this enzyme.  A sheet of X-ray film is then placed next to the membrane, which allows visualization of individual proteins. In a variation of the technique, an unlabeled first antibody is used to bind the protein on the membrane, and a second antibody, directed against the first antibody, is used for detection.

The main advantage of western analysis is that it does not require isotopic labeling of proteins and can be used with tissues and organs, as well as cultured cells.

A variation of the western blot is used to identify antibodies to human immunodeficiency virus in clinical specimens and donated blood. Viral proteins are fractionated by electrophoresis and transferred to a membrane as describe above. The membrane is then incubated with the clinical sample. If antibodies against HIV are present, they will react with one or more of the viral proteins on the membrane. Such an assay is being used to estimate the extent of infection with the retrovirus XMRV in the general population.

Note that the w in western blot is not capitalized. The n of northern analysis (a method in which RNA is detected on a thin membrane) is also lower case. However, Southern analysis deserves a capital S – it’s the last name of Edwin Southern, who in 1975 developed the technique for detecting DNA immobilized on a membrane.