Study of the time of synthesis and nature of viral proteins in the infected cell requires the ability to distinguish virus-encoded proteins in a background of cellular ones, and to fractionate such viral proteins away from cellular components of the infected cell. Given the large amount of mass of the biological macromolecules contained in the cell, the process of viral protein or nucleic acid purification can be difficult and requires technical ingenuity.
Although the detection of viral proteins against the background of cellular material is difficult, the task is made somewhat more tractable in many virus infections because the infection leads to a partial or total shutoff of host cell mRNA or protein synthesis while viral proteins and mRNA are synthesized at high rates. This means that if radioactive amino acids are added to infected cells to serve as precursors to protein synthesis, they will be preferentially incorporated into viral products. In such a situation, the addition of radioactive precursors for a short period at a specific time after infection (a pulse of radioactive precursors), followed by isolation of total cellular material, will yield a mix of both viral and cellular material, but only the viral material will have incorporated significant amounts of radioactivity. Thus, size fractionation of the proteins in the infected cell provides a biochemical "snapshot" of whichever proteins are being synthesized at the time of labeling.
It is very important to remember that virus infection often leads to increased expression of some host cell proteins as part of its defenses (see Chapter 10). Therefore, the profile of proteins synthesized in a cell infected even with a virus that is extremely efficient in inhibiting host functions will not necessarily contain only viral products. Also, infections by some very important viruses do not result in efficient shutoff of host protein synthesis — in such a case, the proteins labeled in a pulse will be a mixture of cellular and viral proteins.
Examples of pulse labeling experiments following infections with some viruses that do shutoff host protein synthesis are shown in Fig. 12.1. For the left panel, radiolabeled amino acids were added to poliovirus-infected cells at the time after infection shown, and then proteins were fractionated. Many of the bands of radioactivity seen by exposing the gel to x-ray film are the result of the expression of viral proteins. Some of the more notable ones are indicated, as are some cellular proteins.
Several features of this pattern of pulse labeling are readily apparent. First, the amount of the capsid protein VP2 does not appear equimolar with that of VP1 and VP3, as was seen in the fractionation of proteins found in the mature capsid shown in Fig. 11.4. The reason for this is that VP2 is derived from the processing of VP0, and therefore, some of the radioactivity that would be in the peak of VP2 is actually in the VP0 band.
Another feature is that the viral proteins indicated are in the same relative proportions at all times measured. As described in Chapter 14, poliovirus infection is characterized by the expression of only one mRNA molecule and all proteins are derived from a large precursor that cannot be seen in this gel. However, portions of precursor proteins such as 3CD are clearly seen.
A third feature of the gel can be seen in examination of the cellular proteins labeled after infection. Although the synthesis of some is clearly shutoff, the synthesis of others persists. This is an example of the fact that some cellular genes continue to be expressed (or can be induced) following infection.
The effect of an HSV-1 infection on total protein synthesis in infected cells is shown in the right panel of Fig. 12.1. It is evident that the pattern of labeled viral proteins changes markedly with time. Some viral proteins synthesized at 3 hours following infection are no longer synthesized at later times. Conversely, some proteins are only labeled at later times after infection.
Fig. 12.1 Changes in the proteins synthesized in virus-infected cells with time after infection. The left panel shows an experiment in which HeLa cells were infected with the Sabin (vaccine) strain of poliovirus, and labeled with 35S-labeled methionine for 2-hour pulses at the times (hours post-infection) shown at the top of the gel. Protein was isolated and then fractionated on a denaturing gel, and radioactive proteins were localized by autoradiography (exposure to x-ray film). The capsid proteins are indicated, as are other nonstructural poliovirus-encoded proteins. Some cellular proteins whose synthesis is shutoff following infection are shown with the letter "O," while a couple whose synthesis continues is indicated by "O*." (Photograph courtesy of S. Stewart and B. Semler.) The right panel shows a similar experiment carried out by labeling HSV-1-infected Vero cells for 30-minute periods at the times shown after infection. Some cellular proteins that are rapidly shutoff are indicated with "C." "C*" marks proteins that do not appear to be shutoff or whose synthesis increases for a period following infection. Viral proteins synthesized early after infection are indicated by "E." Note that there are at least two subsets, E1 and E2, which differ in the length of time that their synthesis continues. Similarly, there are at least two subsets of late proteins ("L"); some are clearly synthesized at the earliest times while others are only synthesized later. In both panels mock-infected cells (M) show the patterns of proteins synthesized in uninfected cells. (Photograph courtesy of S. Silverstein.)
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