Principles of Virology, Volume 1. Jane Flint

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.

Perspectives

      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 stages of the reproduction cycle in a simple and quantitative fashion. With modest expenditure of time and reagents, virologists can deduce a great deal about viral translation, genome replication, and assembly. It has long been assumed from such one-step growth analyses that the same steps of the viral reproduction cycle occur at the same time in every infected cell. However, results from analyses of single infected cells demonstrate that the same steps can take place at vastly different times in individual cells in the population. We now understand that results from population-based studies of viral reproduction comprise an average of events occurring in individual cells. One-step growth analyses with single cells have the potential of unraveling the viral and cellular basis for such individual heterogeneity.

Figure 2.22 Single-cell virology. (A) A microfluidic device with 6,400 wells is fitted with four separate sample inlets (green) and pneumatic control lines (red) that permit each well to be sealed and isolated. A small part of the device is magnified at the top, showing an array of 24 wells, and four wells are further magnified to the left. (B) The device can be used to measure real-time fluorescence in cells infected with a virus encoding a fluorescent reporter. The production of fluorescence is shown in the graph and illustrated in the views of single cells in individual cells above. There is a lag in the detection of fluorescence in an infected cell (t_i), followed by virus reproduction (t_j) and a decline in fluorescence caused by cell lysis (t_k). Reprinted from Guo F et al. 2017. Cell Rep 21:1692-1704, with permission.

      From the humble beginnings of the one-step growth curve, many new methods have propelled our understanding of viruses and infected cells to greater depths and at unprecedented speed. An astounding array of technologies, including high-throughput sequencing, proteomics, and single-cell approaches, have been developed. These methods have already led to significant discoveries about viral evolution, reproduction, and pathogenesis. We are truly in a remarkable era, when few experimental questions are beyond the reach of the techniques that are currently available.

REFERENCES

       Books

      Green MR, Sambrook J. 2012. Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

      Maramorosch K, Koprowski H (ed). 1968. Methods in Virology. Academic Press, New York, NY.

       A series of volumes begun in the 1960s that contain review articles on classic virological methods.

       Review Articles

      Dulbecco R, Vogt M. 1953. Some problems of animal virology as studied by the plaque technique. Cold Spring Harb Symp Quant Biol 18:273–279.

      Kuadkitkan A, Wikan N, Smith DR. 2016. Induced pluripotent stem cells: a new addition to the virologists armamentarium. J Virol Methods 235:191–195.

      Ramani S, Crawford SE, Blutt SE, Estes MK. 2018. Human organoid cultures: transformative new tools for human virus studies. Curr Opin Virol 29:79–86.

      van Dijk EL, Jaszczyszyn Y, Naquin D, Thermes C. 2018. The third revolution in sequencing technology. Trends Genet 34:666–681.

      Wang I-H, Burckhardt CJ, Yakimovich A, Greber UF. 2018. Imaging, tracking and computational analyses of virus entry and egress with the cytoskeleton. Viruses 10:1–29.

       Papers of Special Interest

      Ellis EL, Delbrück M. 1939. The growth of bacteriophage. J Gen Physiol 22:365–384.

       A landmark paper describing the development of the plaque assay to measure infectivity of bacteriophage and its use in studying viral reproduction by one-step analysis.

      Reed LJ, Muench H. 1938. A simple method