Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 112

Understanding the roles of both viral and cellular proteins at various stages of viral reproduction is essential for elucidating molecular mechanisms and for developing strategies for blocking pathogenic infections. As viral genomes have a limited set of genes, the viral proteins or genetic elements that are essential at each step can be deduced by introducing mutations and observing phenotypes. Identifying critical cellular genes begins with the identification of cellular proteins that are included in virus particles and/or bind to viral proteins (in vitro or in cells).

Once candidates are identified, the contribution of the cellular protein to viral reproduction may be evaluated by observing the effects of

 specific small-molecule inhibitors of the protein’s function (inhibitory drugs)

 synthesis of an altered protein, known to have a dominant negative effect on its normal function

 treatment with small RNAs that induce mRNA degradation (see Chapter 10) and reduce the concentration of the cellular protein

 reproduction in cells in which the candidate gene has been mutated or deleted

Even after applying the multiple approaches and methods described above, identifying relevant cellular proteins and evaluating their roles in viral reproduction is seldom easy. The problems encountered include the following.

 More than one protein may provide the required function (redundancy).

 The function of the protein might be essential to the cell, and mutation of the gene that encodes it (or inhibition of protein production) could be lethal.

 Only small quantities of the protein might be required, and reducing its activity with an inhibitor, or its concentration may be insufficient to induce a defect in viral reproduction.

 The cellular protein might provide a slight enhancement to viral reproduction that could be difficult to detect but may be physiologically significant.

 Synthesis of an altered cellular gene or overexpression of a normal cellular gene may produce changes that affect virus reproduction for reasons that are irrelevant to the natural infection (artifacts).

Given these difficulties, it is not surprising that the literature in this area is sometimes contradictory and the results can be controversial.

Infection of single cells with vesicular stomatitis virus identified 496 mutations that arose in 24 hours during genome replication within 90 cells. The rates of mutation varied among individual cells, and this high value represents an average for all of the cells. In addition, preexisting viral genetic diversity was used to track infection in single cells. These investigations revealed that even though viruses were added at a low multiplicity of infection, most cells had acquired more than one virus particle. The results suggested that virus particles have a tendency to stick to one another, raising further challenges to determining multiplicities of infection.

Single-cell studies have demonstrated that measurements of virus reproduction in populations of cells do not represent the diversity that exists among individual cells. Consequently, they will likely become a complementary tool to the one-step growth experiment for studying virus infection.