Principles of Virology. Jane Flint

required for retroviral vectors are 5′- and 3′-terminal sequences (yellow and blue, respectively) that control gene expression and packaging of the RNA genome. The foreign gene (blue) and promoter (green) are inserted between the viral sequences. To package this DNA into viral particles, it is introduced into cultured cells with plasmids that encode viral proteins required for encapsidation, under the control of a heterologous promoter and containing no viral regulatory sequences. No wild-type viral RNA is present in these cells. If these plasmids are introduced alone, virus particles that do not contain viral genomes are produced. When all three plasmids are introduced into cells, retrovirus particles that contain only the recombinant vector genome are formed. The host range of the recombinant vector can be controlled by the type of envelope protein. Envelope protein from amphotropic retroviruses allows the recombinant virus to infect human and mouse cells. The vesicular stomatitis virus glycoprotein G allows infection of a broad range of cell types in many species and also permits concentration with simple methods.

Perspectives

      The information presented in this chapter can be used for navigating this book and for planning a virology course. Figures 3.1 to 3.7 illustrate seven strategies based on viral mRNA synthesis and genome replication and serve as the points of departure for detailed analyses of the principles of virology. For those who prefer to teach virology based on specific viruses or groups of viruses, the material in this chapter can be used to structure individual reading or to design a virology course while adhering to the overall organization of this textbook by function. Reference to this chapter provides answers to questions about specific virus families. For example, Fig. 3.5 provides information about (+) strand RNA viruses and Fig. 3.10 indicates specific chapters in which these viruses are discussed.

      Since the earliest days of experimental virology, genetic analysis has been essential for studying viral genomes. Initially, methods were developed to produce viral mutants by chemical or UV mutagenesis, followed by screening for readily identifiable phenotypes. Because it was not possible to identify the genetic changes in such mutants, it was difficult to associate proteins with virus-specific processes. This limitation was surmounted with the development of cloned infectious DNA copies of viral genomes, an achievement that enabled the introduction of defined mutations. These methods for reducing or ablating the expression of specific viral or cellular genes comprise a complete genetic toolbox that provides countless possibilities for studying the viral genome and the interaction of viral gene products with those of the cell. The ability to manipulate cloned DNA copies of viral genomes has also enabled the development of viruses as vectors for the expression of foreign genes, for gene therapy, viral oncotherapy, and to deliver vaccines to prevent infectious diseases. How ironic it is that our study of the viruses that cause disease has led to their transformation into therapeutic agents!

REFERENCES

       Review Articles

      Baltimore D. 1971. Expression of animal virus genomes. Bacteriol Rev 35:235–241.

      Knott GJ, Doudna JA. 2018. CRISPR-Cas guides the future of genetic engineering. Science 361:866–869.

      Krupovic M, Dolja VV, Koonin EV. 2019. Origin of viruses: primordial replicators recruiting capsids from hosts. Nat Rev Microbiol 17:449–458.

       Papers of Special Interest

      Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J, Min Jou W, Molemans F, Raeymaekers A, Van den Berghe A, Volckaert G, Ysebaert M. 1976. Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature 260:500–507.

       The first complete genome sequence of any kind.

      Taniguchi T, Palmieri M, Weissmann C. 1978. QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host. Nature 274:223–228.

       The first infectious virus from a cloned DNA copy of a viral genome.

      Crotty S, Cameron CE, Andino R. 2001. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc Natl Acad Sci U S A 98:6895– 6900.

       Only a two-fold increase in poliovirus genome mutations is needed to push the population over the error threshold.

      Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498.

       The first report that synthetic siRNAs can silence gene expression in mammalian cells.

      Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821.

      The Cas9 endonuclease is engineered into a two-component system by fusing the two RNA molecules into a single-guide RNA that, when combined with Cas9, cuts the DNA target specified by the guide RNA.

      Wolf YI, Kazlauskas D, Iranzo J, Lucía-Sanz A, Kuhn JH, Krupovic M, Dolja VV, Koonin EV. 2018. Origins and evolution of the global RNA virome. mBio 9:e02329–18.

       Analysis of RdRp sequences illuminates RNA virus evolution.

STUDY QUESTIONS

      1 The Baltimore scheme is useful for predicting the path from the viral genome to mRNA, but to do this a few other facts are needed. Which of the following is not one of these facts?mRNA cannot be made from ssDNAdsRNA can be directly translated into protein since it contains a (+) strandA gapped dsDNA must be repaired before transcription can beginCells do not produce RNA-dependent RNA polymeraseNone of these are incorrect

      2 Why is mRNA placed at the center of the Baltimore scheme?Because all virus particles contain mRNAThere is no specific reasonBecause all viral genomes are mRNAsBecause mRNA must be made from all viral genomesBecause Baltimore studied mRNA

      3 Which DNA genome, on entry into the cell, can be immediately copied into mRNA?dsDNAGapped dsDNACircular ssDNALinear ssDNAAll of the above

      4 Which statement about viral RNA genomes is correct?(+) ssRNA genomes may be translated to make viral proteindsRNA genomes can be directly translated to make viral protein(+) ssRNA virus replication cycles do not require a (–) strand intermediateRNA genomes can be copied by host cell RNA-dependent RNA polymerasesAll of the above

      5 Will viral RNA extracted from virus particles of a virus with a (–) ssRNA genome initiate an infection after transfection into a permissive cell? Into a susceptible cell? Explain your answers.

      6 Why must all RNA virus genomes encode an RNA-dependent RNA polymerase?

      7 Viruses with segmented RNA genomes can undergo a process that viruses with unimolecular RNA genomes cannot. What is this process called and how does it occur?

      8 This is the genome of a (–) strand RNA virus. It is 14 kb in length.If this purified RNA is introduced into cultured cells by transfection, will infectious viruses be produced? Why or why not?Describe two different strategies for producing seven different viral polypeptides from this genome.

      9 You infect two plates of cells with virus at multiplicities of infection (MOI) of 1 and 10. After 4 h of incubation,