John Wiley & Sons - 2004 - Analysis of Genes and Genomes

of foreign DNA. Even with the replacement of existing genes, the maximum foreign DNA insert size is limited to 2300 bp (Naim and Roth, 1994).

12.3.2Adenovirus

Adenoviruses are responsible for approximately 25 per cent of the ‘common colds’ that we suffer. There are many distinct types that can cause infections in humans, but they are all non-enveloped icosohedral viruses (ranging from 60 to 90 nm long) that contain linear double-stranded DNA genomes 36 kbp in size (Figure 12.4). The transcription of the adenovirus genome occurs

(a)

Figure 12.4. Adenovirus. (a) Electron micrograph of a human faecal adenovirus. Image courtesy of Tony Oliver (IBMS London Region Virology Discussion Group). (b) The adenoviral genome. The approximate location of some of the early (E) and late (L) genes are shown, along with the site of the major late promoter (MLP), and an approximately 300 bp packaging site ( ), which is essential for the packaging of viral genome into the assembled capsid. Both ends of the genome are composed of 100 bp inverted terminal repeats (ITRs). ITRs serve as origins of DNA replication by priming the replication process and acting as assembly points for the proteins of the replication complex. The adenovirus genome also encodes several viral associated RNAs (VA RNAs) that are transcribed constitutively at a high level using RNA polymerase III. VA RNAs are 160 nucleotides in length and have a high GC content and a high degree of secondary structure. They may serve to regulate mRNA splicing or to control of the rate of translation of late genes (Mathews, 1995)

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in two phases – early and late – occurring either before or after virus DNA replication, respectively. Transcription is accompanied by a complex series of splicing events, with four early regions of gene transcription (E1 –E4, which are required for viral replication), and an MLP producing the late genes (L1 –L5, which produce the viral capsid) (Logan and Shenk, 1984). The virus will replicate in many different mammalian cell lines when the naked genomic DNA is transfected into them. Additionally, the virus produces large numbers of progeny (up to 105 virions per infected cell), which means that viral particles, recombinant or otherwise, can be purified in large amounts with ease.

Most vectors derived from the adenoviral genome are replication deficient. They lack the essential E1 genes and often the non-essential E3 gene. Since they are replication defective, they must be propagated in cell lines that have also been transfected with DNA containing the E1 genes – e.g. the human embryonic kidney cell line 293 – to supply E1 function in trans (Graham et al., 1977). This type of vector has a maximum capacity for foreign DNA of about 7 kbp. Other adenoviral vectors lack either the E2 or E4 genes in addition to E1 and E3 to increase the capacity to carry foreign DNA, these functions again, being supplied in trans from the cell line (Gorziglia et al., 1996).

Adenoviruses bearing foreign DNA can be used to produce the foreign protein in many different cell types, but gene expression is usually transient because the viral DNA does not integrate into the host genome. The lack of integration may, however, be advantageous if adenoviral vectors are used in gene therapy trails (see below). Additionally, tissue specific gene expression is possible with adenoviral vectors if the foreign gene is placed under the control of cell-specific promoter and enhancer elements, e.g. the myosin light chain 1 promoter (Shi, Wang and Worton, 1997) or the smooth muscle cell SM22a promoter (Kim et al., 1997), or by direct delivery to a local area in vivo (Rome et al., 1994).

Adenoviral vectors are useful because they are highly efficient at getting DNA into cells. They are capable of containing DNA inserts up to about 8 kbp in size and they can infect both replicating and differentiated cells. Additionally, since they do not integrate into the host genome, they cannot bring about mutagenic effects caused by random integration events. The disadvantages of adenoviral vectors include the following.

•Expression is transient since the viral DNA does not integrate into the host.

•Adenoviral vectors are based on an extremely common human pathogen and in vivo delivery may be hampered by prior host immune response to one type of virus.

12.3.3Adeno-associated Virus (AAV)

Adeno-associated virus was first discovered as a contaminant of adenovirus preparations (Atchison et al., 1965). Despite its name, AAV is not related to adenovirus but rather it is a member of the parvovirus family, which has a single-stranded DNA genome approximately 5000 nucleotides in size. The virus is commonly found in human tonsil tissue, although no specific disease of man appears to be associated with it. AAVs are naturally replication deficient, and require the presence of another virus – e.g. adenovirus – in order to complete their own replicative cycle. Some of the early adenovirus genes are required to promote AAV replication. However, the treatment of AAV infected cells with ultraviolet light, cycloheximide or some carcinogens can replace the requirement for helper virus (Bantel-Schaal, 1991). Therefore the requirement for a helper virus appears to be for a modification of the cellular environment rather than a specific viral protein.

In the absence of helper virus, the AAV genome integrates into the host cell, where it remains as a latent provirus. The integration of the viral DNA into the host genome is not, however, a random process; it specifically integrates into the same genetic location on chromosome 19 (Kotin et al., 1990). AAV based vectors exploit two 145-base ITR sequences found within the viral genome, which are the only DNA elements required for replication, transcription, provirus integration and rescue. These repeats form hair-pin loop structures that are essential for viral replication. Cloning into the AAV genome is facilitated by inserting the inverted terminal repeats into a plasmid vector and then cloning foreign DNA between them. Linear DNA fragments containing the foreign gene flanked by the ITRs may be prepared from such plasmids following digestion with restriction enzymes. This DNA is then transfected into cells, along with helper plasmid to supply the AAV genes needed for viral integration. Viral stocks may then be prepared by infecting the cells with adenovirus to stimulate AAV lytic replication and packaging. Finally, the AAV viral particles may purified from the contaminating adenovirus using biochemical methods (Gao et al., 2000). This type of approach suffers from the use of adenovirus to recover the AAV particles. The likelihood of contamination is high and therefore the recombinant AAV produced cannot be used for human gene therapy trials. An alternative approach to the production of recombinant AAV particles is shown in Figure 12.5. Here, the foreign gene flanked by the ITRs is co-transfected into human 293 kidney cells together with two other DNA fragments – one containing the genes required for the production of single-stranded AAV genomic DNA, and the other bearing the adenoviral genes required for lytic replication and packaging (Matsushita et al., 1998). The recombinant AAV

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Partial uncoating of the particle occurs prior to the reverse transcriptase enzyme converting the RNA genome into a linear double-stranded DNA copy. DNA synthesis initiates from a specific tRNA molecule (Mak and Kleiman, 1997) acquired from the host cell in which the virus was produced, which, like the reverse transcriptase, is packaged in the viral particle itself. The newly formed proviral DNA then integrates randomly into the host genome where the viral genes are expressed to make new viral proteins and results in the assembly of new viral particles. The production of new viruses does not directly result in host cell death, but rather the newly formed particles bud from the cell surface. The diseases associated with retroviral infections are usually a consequence of the site of retroviral insertion into the host genome, or as a result of alterations made to the types of cell they infect, e.g. the invasion of the immune system by HIV-1 leading to AIDS.

The genome of a retrovirus is composed of two identical single-stranded RNA molecules (between 8000 and 11 000 nucleotides in length) that, similar to mRNA molecules, each possess a 5 cap and a 3 polyA tail (Ratner et al., 1985). The viral RNA genome cannot be used as a translation template prior to integration into the host. Transcription and subsequent translation only occurs after the DNA version of the genome has been integrated into the host. All retroviruses contain three major structural genes, each of which encodes a number of protein activities (Katz and Skalka, 1994):

•gag – encodes a variety of capsid proteins;

•pol – encodes the reverse transcriptase required to convert the RNA genome into a DNA copy, the integrase needed for the insertion of the DNA into the host genome and sometimes a protease (pro) required for cleavage of the gag gene product during maturation;

• env – encodes the surface and transmembrane proteins found in the envelope.

Some retroviruses also contain other genes; e.g. pro may be encoded by a separate gene. All of the proteins encoded by gag, pol and pro are expressed from a single full-length genomic RNA transcript. The env protein is produced from a spliced mRNA. To compress this number of genes into a small genome, the virus utilizes a number of strategies such as splicing and ribosomal frameshifting (Farabaugh, 1996). For example, the pro gene often overlaps gag and/or pol but is still produced from the same mRNA molecule. The retroviral structural genes are flanked within the genome by LTRs, which become duplicated as part of the replication process prior to integration into the host

genome. Viral RNA synthesis initiates from a single promoter within the lefthand LTR and ends at a polyadenylation signal within the right-hand LTR. Packaging of the transcribed RNA into viral particles requires a packaging signal ( ) located between the left-hand LTR and the structural genes (Figure 12.6(a)).

Packaging constraints on the amount of additional nucleic acid that can maintained within the viral genome mean that most vectors based on retroviruses are usually replication defective. The LTRs, the packaging site ( ) and the primer binding sites are the only genomic elements required for the replication process. Therefore, as we discussed for adenoviral vectors above, infective viral particles must be produced in specially constructed cell lines that can provide the necessary viral proteins. For example, a foreign gene may be cloned into a plasmid that contains DNA versions of the elements described above such that it is located between the leftand right-hand LTRs. The recombinant viral shuttle vector is then transfected into a cell line that constitutively expresses the viral reverse transcriptase and capsid proteins. Viruses produced from this cell line can then be used to infect the target cells, where the vector will become integrated into the genome and the foreign gene expressed. In addition to the foreign gene, a marker gene must also be used to assess whether an individual cell has taken up the recombinant DNA.

The major advantages to using retroviral based vectors arise from the stability of the integration of the viral genome into the host. The integration of a single copy of the viral DNA at a random location within the host’s genome allows for the long-term expression of the integrated foreign gene. Additionally, retroviruses represent a highly efficient mechanism for the transfer of DNA into cells. The disadvantages of such vectors are the random nature of the integration process, which may have deleterious effects on the host cell, and the general requirement that retroviruses have to infect only dividing cells. This may severely limit their use. Only lentiviruses, e.g. HIV1, will also infect and replicate in non-dividing cells. Therefore, much effort has been directed into making suitably safe lentivirus vectors (Zufferey et al., 1998).

12.4Selectable Markers and Gene Amplification in Animal Cells

Like all of the other DNA transfer processes we have discussed, the incorporation of foreign DNA into animal cells needs to be accompanied by a phenotypic change to distinguish the transfected cells from those that have not taken up the foreign DNA (Figure 12.7). Some of the first experiments to identify transfected animal cells involved the complementation of a nutritional defect in a cell line. For example, human cells defective in the gene encoding the enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT; part of

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Figure 12.7. The structure of the bleomycin–bleomycin binding protein complex (Maruyama et al., 2001). Binding sites for the drug (shown in red) are located in deep clefts in the sides of the dimeric protein (green and blue)

the inosinate cycle for the salvage of purine bases and the production of inosine monophosphate) and therefore unable to grow in medium containing hypoxanthine, aminopterin and thymidine (HAT, which blocks de novo inosine monophosphate production (Lester et al., 1980)) were transfected with total genomic DNA from a wild-type cell line. Very rarely, cells could be isolated that were able to grow in this medium, indicating that they had acquired the ability to make HPRT from the wild-type cells (Szybalska and Szybalski, 1962). Similarly, mouse cells deficient in the enzyme thymidine kinase (TK, which is part of the nucleotide biosynthesis salvage pathway) are unable to grow on HAT medium, but can be transfected with the herpes simplex virus (HSV) Tk gene to allow growth on this medium (Wigler et al., 1977). A number of other such metabolic markers have also been used to monitor the transfection process, but they all suffer from the requirement of a mutant cell line in order to detect transformed cells. This need has been overcome by using dominant selectable markers that confer a drug resistance phenotype to the transfected cells (Table 12.1). Antibiotics such as ampicillin have no effect on eukaryotic cells due to the lack of an animal cell wall. Some other antibiotics, particularly those that are protein synthesis inhibitors, are active against both prokaryotes and eukaryotes. For example, the E. coli transposon Tn5 encodes the kanamycin-resistance gene (Yamamoto and Yokota, 1980). Kanamycin is an aminoglycoside antibiotic that interferes with translation and induces bacterial cell death through site-specific targeting of ribosomal 16S RNA. At higher concentrations, aminoglycosides also inhibit protein synthesis in mammalian cells probably through non-specific binding to eukaryotic ribosomes and/or nucleic acids (Mingeot-Leclercq, Glupczynski and Tulkens, 1999). To achieve resistance as a method of selection, another gene encoded within the Tn5 transposon (producing neomycin phosphotransferase) is placed under the

Table 12.1. Markers for the selection of DNA fragments added to higher eukaryotic cells

control of a mammalian promoter, e.g. the HSV Tk promoter or the SV40 early promoter (Berg, 1981). We will discuss promoters used to drive expression of genes in animal cells in more detail below.

The exposure of animal cells to high levels of toxic drugs for prolonged periods for the purposes of recombinant selection can give rise, at a very low frequency, to the formation of cells that have become spontaneously highly resistant to the drug. For example, mouse cells exposed to methotrexate (a folic acid analogue that is an inhibitor of the enzyme dihydrofolate reductase, DHFR) have been shown to undergo three types of mutation to generate resistance (Schimke et al., 1978):

•mutations within the DHFR enzyme to generate inhibitor resistance,

•mutations that prevent cellular uptake of the drug and