Creation of Camouflaged Viruses

CREATION OF CAMOUFLAGED VIRUSES

Another worrying achievement of genetic engineering is the ability to hide a potentially dangerous virus inside a harmless bacterium. This is not really novel, because bacteriophages naturally insert their genomes into bacterial chromosomes or plasmids and later reemerge to infect other hosts. Using standard molecular biology, it is possible to clone the entire genome of a small virus that normally infects animals or plants and then insert it into a bacterial plasmid, essentially hiding a pathogenic virus inside harmless bacteria. To accommodate the genomes of larger viruses, bacterial artificial chromosomes may be used instead of plasmids. In the case of RNA viruses, a cDNA copy of the virus genome must first be generated by reverse transcriptase before cloning it into a bacterial vector. There are three main technical issues to face when cloning complete virus genomes:

(a) The fidelity of reverse transcriptase and of the polymerase used during PCR. The enzymes that were originally available introduced too many errors. Nowadays, high-fidelity reverse transcriptase and PCR polymerases are available. Hence the length of RNA or DNA that can be generated error-free has greatly increased.

(b) Suitable vectors to carry large inserts of DNA. Vectors able to carry extremely large inserts, such as bacterial or yeast artificial chromosomes (BACs and YACs), have been developed to clone and sequence large segments of eukaryotic genomes.

(c) Certain base sequences found in virus genomes are not stably maintained or replicated on plasmids in bacterial hosts. These are referred to as poison sequences . For example, the cDNA version of yellow fever virus could not be cloned in one piece. Instead it was cloned as two segments that were replicated separately in a bacterial host. To generate a complete, functional cDNA, the two fragments had to be ligated invitro . This problem can sometimes be solved by using a suitable low-copy vector.

Many cell types, both bacterial and eukaryotic, can take up DNA or RNA under certaincircumstances by transformation. Consequently the naked nucleic acidgenomes of many viruses (both DNA and RNA) are infectious even in the absence of theirprotein capsids. Once a virus genome is cloned, DNA molecules containing the virus genomecan be generated by replication of the plasmid inside the bacterial host cell. Although suchDNA contains extra plasmid sequences, it still may be infectious if transformed into theappropriate host cell.

Amazingly, this is sometimes even true for RNA viruses—that is, the cDNA version of an RNA virus can successfully infect host cells and give rise to a new crop of RNA-containing virus particles. For this to occur, the cDNA must enter the nucleus of the host cell and be transcribed to give an RNA copy of the virus. The viral RNA then proceeds through its normal replication cycle. This has been demonstrated for RNA viruses such as poliovirus, influenza, and coronavirus (one of the causative agents of common colds).

An improved strategy for generating RNA viruses is to clone the cDNA version of their genomes onto a bacterial plasmid downstream of a strong promoter ( Fig. 23.13 ). In this case, the natural RNA version of the virus genome will be generated by transcription. This may be done inside the bacterial host cell by using a bacterial promoter. Alternatively, a eukaryotic promoter may be used to improve transcription of cDNA into viral RNA once the cDNA has entered the eukaryotic host cell. The technology thus exists to create bacteria carrying “hidden” plasmid-borne animal viruses. If the complete cDNA from an RNA virus is placed under control of a strong bacterial promoter, the bacterial cell could generate large amounts of infectious viral RNA internally by transcription. When the bacterial cell dies and disintegrates, the viral RNA would be liberated. If a dangerous human RNA virus was loaded into a harmless intestinal bacterium under control of a promoter designed to respond to conditions inside the intestine, this could pose a serious threat.