has been increasing interest in these vectors to target therapeutic genes to smooth muscle and other differentiated tissues, which are highly susceptible and support sustained high-level expression of foreign genes. Although the first-generation adenovirus-associated virus vectors were limited in the size of inserts that could be transferred, other systems have been developed to overcome the limited genetic capacity (Fig. 3.15). The cell specificity of adenovirus-associated virus vectors has been altered by inserting receptor-specific ligands into the capsid. In addition, many new viral serotypes that vary in their tropism and ability to trigger immune responses have been identified or generated.
Vaccinia virus and other animal poxvirus vectors offer the advantages of a wide host range, a genome that accepts very large fragments, high expression of foreign genes, and relative ease of preparation. Foreign DNA is usually inserted into the viral genome by homologous recombination, using an approach similar to that described for marker transfer. Because of the relatively low pathogenicity of the virus, poxvirus recombinants have been considered candidates for human and animal vaccines.
Baculoviruses, which infect arthropods, have large circular dsDNA genomes. These viruses have been modified to become versatile and powerful vectors for the production of proteins for research and clinical use. The general approach is to replace the viral polyhedron gene with the gene of interest. Recombinant viruses are produced in E. coli using a bacmid vector that harbors the baculovirus genome. The gene to be introduced is inserted into the baculovirus genome by recombination. Strong viral promoters are used to obtain high levels of protein production. Recombinant baculoviruses are obtained after transfection of bacmids into insect cells and have been used for protein production for research purposes and for large-scale synthesis for commercial uses. Examples include the influenza virus vaccine FluBlok, which consists of the viral HA proteins produced in insect cells via a baculovirus vector, and porcine circovirus 2 vaccine for the prevention of fatal disease in swine.
Figure 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
