Characterization of viral mRNA expressed during infection

Viral mRNA expressed during infection also can be analyzed and characterized using gel electrophoresis for size fractionation followed by nucleic acid hybridization. Without hybridization, detection of viral mRNA against the background of cellular RNA is difficult because individual mRNA molecules are not present in high abundance.

Start

Density

Spin to equilibrium

Density

Spin to equilibrium

c ; ; ;

(3)

->

(2)

(1)

Fig. 12.8 Separation of HSV DNA from cellular DNA based on differences in base composition. The percentage of G+C residues in a given fragment of dsDNA will determine its buoyant density in CsCl. In the experiment shown, three DNA samples were mixed with a CsCl solution. One sample has a very high G+C content and serves as a density marker. HSV DNA has a lower density, but is significantly higher in G+C content than cellular DNA (approximately 67% versus 48%). For this reason it has a greater buoyant density in an equilibrium gradient of CsCl. Unlike the equilibrium sucrose gradient shown in Fig. 11.1, CsCl solutions are so dense that the gradient will form under the centrifugal force available in an ultracentrifuge. Therefore, the mixture of DNA and CsCl is made and placed in a centrifuge rotor, and the mixture is allowed to form a density gradient by high-speed centrifugation. Following equilibrium, the various DNA fragments can be isolated by careful dropwise collection of the gradient. The graph shows the position of the three DNA species at equilibrium.

Hybridization requires a DNA or RNA probe that is complementary to the mRNA sequences to be detected. Such probes can be prepared readily by use of molecular cloning of viral DNA fragments in bacteria and one of a number of methods for making radioactive probe. This use of recombinant DNA technology provides a convenient and inexpensive source of pure material in large quantities. Some basic methods for cloning viral DNA fragments are briefly outlined in Part V. One of the most important things to remember about nucleic acid hybridization or annealing is that under the proper conditions, the presence of large (even overwhelming) amounts of RNA or DNA with sequences different from that of the test DNA or RNA has no effect on the rate or amount of hybrid formed. This makes nucleic acid hybridization an exquisitely sensitive method for detecting RNA and DNA, and much understanding of the regulatory processes involved in viral and cellular gene expression has a basis in the ability to precisely measure such expression at any given time during the viral replication cycle.

An experiment showing the different mRNAs expressed from two regions of the HSV genome is described in Fig. 12.9. In this experiment, mRNA was isolated from HSV-infected cells at 6 hours after infection. Aliquots then were fractionated by gel electrophoresis and blotted onto a membrane filter. Replicate blots were hybridized with radioactive total viral DNA probe, or with a probe made from cloned DNA fragments from specific regions of the viral genome (as shown).

In another experiment also shown in Fig. 12.9(d), HSV mRNA was isolated at two different times (3 and 8 hours) following infection. At 3 hours, viral DNA replication has not yet begun. At 6 hours after infection, it is taking place at a high rate. The two RNA samples were fraction-

HSV genome

Fig. 12.9 Different viral mRNA molecules are encoded by different regions of a viral genome. The diagram shows the 150,000-base-pair HSV genome and the location of three cloned DNA fragments that can be used to hybridize to total infected cell RNA. More detailed information concerning the HSV genome and specific genes can be found in Chapter 17, Part IV. A number of fractionation gels are shown. (a) The total viral mRNA species expressed at 5 hours following infection. The RNA was isolated and fractionated, and a northern blot made of the RNA. This was hybridized with radioactive viral DNA to locate the viral mRNA species. (b) The RNA species expressed in region 1 by hybridization with radioactive DNA from this region only. (c) The different RNAs seen with a probe for region 2. (d) The RNA expressed from region 2 changes in character between 3 and 8 hours following infection (at the intermediate time shown in c, all species are being expressed). The lanes marked "SS" contain radioactive ribosomal RNA included as a size standard.

Total HSV probe

RNA hybridized with cloned fragment 2

I SS

TR RNA hybridized with cloned fragment 1 \ SS

cloned fragment 1 \ SS

(d) Cloned fragment 2 hybridized with RNA isolated at:

8 hr

3 hr post-infection

ated by gel electrophoresis, subjected to northern blotting, and then hybridized with a fragment of radioactive DNA from a specific region of the HSV genome. One can see that the amount of RNA present at the 3-hour time point (early mRNA) is much reduced by 6 hours, and new - late mRNA - is present at this later time.

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