Principles of Virology. Jane Flint

Principles of Virology - Jane Flint


Скачать книгу
proteins. This method was used to describe the virus-host interactome of two herpesviruses.

      While these methods allow definition of virus-cell interactomes, they are not unambiguous. For at least one virus, interactomes determined in different laboratories are very diverse. Most importantly, the observation of a protein-protein interaction does not confirm biological relevance: the roles of such interactions in viral reproduction must be determined by other means (Box 2.12).

      Much of virology research is carried out by using populations of cells in culture or in animals. However, as discovered by virologists in the 1950s, individual cells of the same type can behave very differently with respect to susceptibility and permissiveness to infection and the kinetics of virus production.

      As early efforts to study virus infections in single cells were hampered by technical difficulties, the field failed to progress. This situation has changed with the development of flow cytometry and microfluidics and the adaptation of highthroughput methods, such as genome sequencing and mass spectrometry, to single cells.

      Initially, micropipettes were used to aspirate a single cell at a time from a population, using a microscope. This labor-intensive method was supplanted by fluorescence-activated cell sorting to allow isolation of up to millions of cells in a few hours, according to size, morphology, or synthesis of specific proteins. More recently, automated microfluidic devices have been developed to allow automated capture of single cells using integrated fluidic circuits. Infection, cell lysis, reverse transcription, and amplification are all performed in these systems before high-throughput sequencing.

      WARNING

       Determining a role for cellular proteins in viral reproduction can be quite difficult

      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).

image

      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.

      One-step growth analysis, while simple, remains a powerful tool for studying virus reproduction. When cells are infected at a high multiplicity of infection, sufficient viral nucleic acid or protein can be isolated to allow a study of their production during the infectious cycle. The ability to synchronize infection is the key to this approach. Many of the experimental results discussed in subsequent chapters of this book were obtained using such one-step growth analysis. The power of this approach is such that it reports on all


Скачать книгу