Principles of Virology, Volume 1. Jane Flint

3.15 Adeno-associated virus vectors. (A) Map of the genome of wild-type adeno-associated virus. The viral DNA is single stranded and flanked by two inverted terminal repeats (ITR); it encodes capsid (blue) and nonstructural (orange) proteins. (B) In one type of vector, the viral genes are replaced with the transgene (pink) and its promoter (yellow) and a poly(A) addition signal (green). These DNAs are introduced into cells that have been engineered to produce capsid proteins, and the vector genome is encapsidated into virus particles. A limitation of this vector structure is that only 4.1 to 4.9 kb of foreign DNA can be packaged efficiently. Ad, adenovirus; rAAV, recombinant adenovirus-associated virus.

       RNA Virus Vectors

      A number of RNA viruses have also been developed as vectors for foreign gene expression (Table 3.1). Vesicular stomatitis virus, a (–) strand RNA virus, has emerged as a candidate for vaccine delivery (e.g., ebolavirus and Zika virus vaccines). For production of vaccines, vesicular stomatitis virus is pseudotyped with glycoproteins from other viruses. For example, to produce an ebolavirus vaccine, the vesicular stomatitis virus glycoprotein gene is substituted with that from ebolavirus. Pseudotyped vesicular stomatitis virus also has applications in the research laboratory: these viruses were used to identify cell receptors in haploid cell lines as described above. The virus is well suited for viral oncotherapy because it reproduces preferentially in tumor cells, and recombinant vesicular stomatitis viruses have been engineered to improve tumor selectivity.

Retroviruses have enjoyed great popularity as vectors (Fig. 3.16) because their infectious cycles include the integration of a dsDNA copy of viral RNA into the cell genome, a topic of Chapter 10. The integrated provirus remains permanently in the cell’s genome and is passed on to progeny during cell division. This feature of retroviral vectors results in permanent modification of the genome of the infected cell. The choice of the envelope glycoprotein carried by retroviral vectors has a significant impact on their tropism. The vesicular stomatitis virus G glycoprotein is often used because it confers a wide tissue tropism. Retrovirus vectors can be targeted to specific cell types by using envelope proteins of other viruses.

      An initial problem encountered with the use of gammaretrovirus vectors (e.g., Moloney murine leukemia virus) is that the DNA of these viruses can be integrated efficiently only in actively dividing cells. Another important limitation of the murine retrovirus vectors is imposed by the phenomenon of gene silencing, which represses foreign gene expression in certain cell types, such as embryonic stem cells. An alternative approach is to use viral vectors that contain sequences from human immunodeficiency virus type 1 or other lentiviruses, which can infect nondividing cells and are less severely affected by gene silencing.

Figure 3.16 Retroviral vectors. The minimal viral sequences 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.