Principles of Virology. Jane Flint
Figure 3.14 Adenovirus vectors. High-capacity adenovirus “gutless” vectors contain only the origin-of-replication-containing inverted terminal repeats (ITR), the packaging signal (blue arrows), the viral E4 transcription unit (red arrow), and the transgene with its promoter. Additional DNA flanking the foreign gene must be inserted to allow packaging of the viral genome (not shown). A helper virus (bottom) is required to package the recombinant vector genome. Two loxP sites for cleavage by the Cre recombinase have been introduced into the adenoviral helper genome (red arrowheads). Infection of cells that produce Cre leads to excision of sequences flanked by the loxP sites so that the helper genome is not packaged.
Adenovirus vectors were originally developed for the treatment of cystic fibrosis because of the tropism of the virus for the respiratory epithelium. Adenovirus can infect terminally differentiated cells, but only transient gene expression is achieved, as infected cells are lysed. Yields of particles are high and these viruses can infect many replicating and non-replicating cell types. In the earliest vectors that were designed, foreign genes were inserted into the E1 and/or E3 regions. As these vectors had limited capacity, genomes with minimal adenovirus sequences have been designed (Fig. 3.14). This strategy allows up to 38 kb of foreign sequence to be introduced into the vector. In addition, elimination of most viral genes reduces cytotoxicity and the host immune response to viral proteins, simplifying multiple immunizations. Considerable efforts have been made to modify the adenovirus capsid to target the vectors to different cell types. For example, the fiber protein, which mediates adenovirus binding to cells, has been altered by insertion of ligands that bind particular cell surface receptors. Such alterations could increase the cell specificity of adenovirus attachment and the efficiency of gene transfer, thereby decreasing the dose of virus that need be administered.
Adenovirus-associated virus has attracted much attention as a vector for gene therapy. This virus requires a helper virus for replication; in its absence the genome remains episomal and persists, in some cases with high levels of expression, in many different tissues. There 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