1.5 VIRUSES CAN BE DEFINED IN CHEMICAL TERMS

The first virus was purified in 1933 by Schlessinger using differential centrifugation. Chemical analysis of the purified bacteriophage showed that

virus in paracrystalline form, and this crystallization of a biological material thought to be alive raised many philosophical questions about the nature

of life. In 1937, Bawden and Pirie extensively purified tobacco mosaic

virus and showed it to be nucleoprotein containing ribonucleic acid (RNA).

Thus virus particles may contain either DNA or RNA. However, at this

time it was not known that nucleic acid constituted genetic material.

The importance of viral nucleic acid

In 1949, Markham and Smith found that preparations of turnip yellow

mosaic virus comprised two types of identically sized spherical particles,

only one of which contained nucleic acid. Significantly, only the particles containing nucleic acid were infectious. A few years later, in 1952,

Hershey and Chase demonstrated the independent functions of viral protein and nucleic acid using the head–tail virus, bacteriophage T2 (Box 1.3).

In another classic experiment, Fraenkel-Conrat and Singer (1957) were

able to confirm by a different means the hereditary role of viral RNA.

Their experiment was based on the earlier discovery that particles of tobacco

mosaic virus can be dissociated into their protein and RNA components,

and then reassembled to give particles which are morphologically mature

and fully infectious (see Chapter 11). When particles of two different strain (differing in the symptoms produced in the host plant) were each disassociated and the RNA of one reassociated with the protein of the other, and vice versa, the properties of the virus which was propagated when the resulting “hybrid” particles were used to infect host plants were always those of the parent virus from which the RNA was derived (Fig. 1.5). The ultimate proof that viral nucleic acid is the genetic material comes from numerous observations that under special circumstances purified viral nucleic acid is capable of initiating infection, albeit with a reduced efficiency. For example, in 1956 Gierer and Schramm, and Fraenkel-Conrat independently showed that the purified RNA of tobacco mosaic virus can be infectious, provided precautions are taken to protect it from inactivation by ribonuclease. An extreme example is the causative agent of potato spindle tuber disease which lacks any protein component and consists solely of RNA. Because such agents have no protein coat, they cannot be called viruses and are referred to as viroids. Synthesis of macromolecules in infected cells Knowing that nucleic acid is the carrier of genetic information, and that only the nucleic acid of bacteriophages enters the cell, it is pertinent to review the events occurring inside the cell. The discovery in 1953, by Wyatt and Cohen, that the DNA of the T-even bacteriophages T2, T4, and T6 contains hydroxymethylcytosine (HMC) instead of cytosine made it possible for Hershey, Dixon, and Chase to examine infected bacteria for the presence of phage-specific DNA at various stages of intracellular growth. DNA was extracted from T2-infected E. coli at different times after the onset of phage growth, and analyzed for its content of HMC. This provided an estimate of the number of phage equivalents of HMC-containing DNA present at any time, based on the total nucleic acid and relative HMC content of the intact T2 phage particle. The results showed that, with T2, synthesis of phage DNA commences about 6 minutes after infection and the amount present then rises sharply, so that by the time the first infectious particles begin to appear 6 minutes later there are 50–80 phage equivalents of HMC. Thereafter, the numbers of phage equivalents of DNA and of infectious particles increase linearly and at the same rate up until lysis, even if lysis is delayed beyond the normal burst time. 12 PART I WHAT IS A VIRUS? Phage labeled with S 35 Phage labeled with P 32 Mix with bacteria Mix with bacteria Blend in Waring blender Blend in Waring blender Centrifuge Centrifuge Supernatant (phage) 75% of radioactivity pellet (cells) 25% of radioactivity Supernatant (phage) 15% of radioactivity Pellet (cells) 85% of radioactivity Fig. 1.4 The Hershey–Chase experiment proving that DNA (labelled with 32P) is the genetic material of bacteriophage T2. Hershey and his co-workers also studied the synthesis of phage protein, which can be distinguished from bacterial protein by its interaction with specific antibodies. During infection of E. coli by T2 phage, protein can be detected about 9 minutes after the onset of the latent period, i.e. after DNA synthesis begins, and before infectious particles appear. A few minutes later there are approximately 30–40 phages inside the cell. Whereas the synthesis of viral protein starts about 9 minutes after the onset of the latent period, it was shown by means of pulse–chase experiments that the uptake of 35S into intracellular protein is constant from the start of infection. A small quantity (a pulse) of 35S (as sulfate) was added to the medium at different times after infection and was followed shortly by a vast excess of unlabelled sulfate (chase) to stop any further incorporation of label. When the pulse was made from the ninth minute onward, the label could be chased into material identifiable by its reaction with antibody (i.e. serologically) as phage coat protein. However, if the pulse was made early in infection, it could be chased into protein but, although this was nonbacterial, it did not react with antibodies to phage structural proteins. This early protein comprises mainly virus-specified enzymes that are concerned with phage replication but are not incorporated into phage particles. The concept of early and late, nonstructural and structural viral proteins is discussed in Part II. These classical experiments are typical only of head–tail phages infecting E. coli under optimum growth conditions. E. coli is normally found in the anaerobic environment of the intestinal tract, and it is doubtful that it grows with its optimal doubling time of 20 minutes under natural conditions. Other bacterial cells grow more slowly than E. coli and their viruses have longer multiplication cycles.