are constructs that assist in the cataloging of viruses). A species name is written in italics with the first word beginning with a capital letter (other words should be capitalized if they are proper nouns). For example, the causative agents of poliomyelitis, poliovirus types 1, 2, and 3, are members of the species Enterovirus C. A virus name should never be italicized, even when it includes the name of a host species or genus, and should be written in lowercase: for example, Sida ciliaris golden mosaic virus. A good exercise would be to see how often we have accidentally violated these rules in this textbook.
Figure 3.11 Reassortment of influenza virus RNA segments. (A) Progeny viruses of cells that are coinfected with two influenza virus strains, L and M, include both parents and viruses that derive RNA segments from them. Recombinant R3 has inherited segment 2 from the L strain and the remaining seven segments from the M strain. (B) 32P-labeled influenza virus RNAs were fractionated in a polyacrylamide gel and detected by autoradiography. Migration differences of parental viral RNAs (M and L) permitted identification of the origin of RNA segments in the progeny virus R3. Panel B reprinted from Racaniello VR, Palese P. 1979. J Virol 29:361–373.
Engineering Mutations into Viral Genomes
Infectious DNA Clones
Recombinant DNA techniques have made it possible to introduce any kind of mutation anywhere in the genome of most animal viruses, whether that genome comprises DNA or RNA. The quintessential tool in virology today is the infectious DNA clone, a dsDNA copy of the viral genome that is carried on a bacterial vector such as a plasmid. Infectious DNA clones, or in vitro transcripts derived from them, can be introduced into cultured cells by transfection (Box 3.8) to recover infectious virus. This approach is a modern validation of the Hershey-Chase experiment described in Chapter 1. The availability of site-specific bacterial restriction endonucleases, DNA ligases, and an array of methods for mutagenesis has made it possible to manipulate these infectious clones at will. Infectious DNA clones also provide a stable repository of the viral genome, a particularly important advantage for vaccine strains. As oligonucleotide synthesis has become more efficient and less costly, the assembly of viral DNA genomes up to 212 kbp has become possible (Box 3.9).
DNA viruses. Current genetic methods for the study of most viruses with DNA genomes are based on the infectivity of viral DNA. When deproteinized viral DNA molecules are introduced into permissive cells by transfection, they generally initiate a complete infectious cycle, although the infectivity (number of plaques per microgram of DNA) may be low. For example, the infectivity of deproteinized human adenoviral DNA is between 10 and 100 PFU per μg. When the genome is isolated by procedures that do not degrade the covalently attached terminal protein, infectivity is increased by 2 orders of magnitude, probably because this protein facilitates the assembly of initiation complexes on the viral origins of replication.
The complete genomes of polyomaviruses, papillomaviruses, and adenoviruses can be cloned in plasmid vectors, and such DNA is infectious under appropriate conditions. The DNA genomes of herpesviruses and poxviruses are too large to insert into conventional bacterial plasmid vectors, but they can be cloned into vectors that accept larger insertions (e.g., cosmids and bacterial artificial chromosomes). The plasmids containing such cloned herpesvirus genomes are infectious. In contrast, poxvirus DNA is not infectious, because the viral promoters cannot be recognized by cellular DNA-dependent RNA polymerase. Poxvirus DNA is infectious when early functions (viral DNA-dependent RNA polymerase and transcription proteins) are provided by complementation with a helper virus.
RNA viruses. (i) (+) strand RNA viruses. The genomic RNA of retroviruses is copied into dsDNA by reverse transcriptase early during infection, a process described in Chapter 10. Such DNA is infectious when introduced into cells, as are molecularly cloned forms inserted into bacterial plasmids.
Infectious DNA clones have been constructed for many (+) strand RNA viruses. An example is the introduction of a plasmid containing cloned poliovirus DNA into cultured mammalian cells, which leads to the production of progeny virus (Fig. 3.12A). The mechanism by which cloned poliovirus DNA initiates infection is not known, but it has been suggested that the DNA enters the nucleus, where it is transcribed by cellular DNA-dependent RNA polymerase from cryptic, promoter-like sequences on the plasmid. The resulting (+) strand RNA transcripts initiate an infectious cycle. During genome replication, the extra terminal nucleotide sequences transcribed from the vector must be removed or ignored, because the virus particles that are produced contain RNA with the authentic 5′ and 3′ termini.
By incorporating promoters for bacteriophage T7 DNA-dependent RNA polymerase in plasmids containing poliovirus DNA, full-length (+) strand RNA transcripts can be synthesized in vitro. The specific infectivity of such RNA transcripts resembles that of genomic RNA (106 PFU per μg), which is higher than that of cloned DNA (103 PFU per μg).
TERMINOLOGY
DNA-mediated transformation and transfection
The introduction of foreign DNA into cells is called DNA-mediated transformation to distinguish it from the oncogenic transformation of cells caused by tumor viruses and other insults. The term “transfection” (transformation-infection) was coined to describe the production of infectious virus after transformation of cells by viral DNA, first demonstrated with bacteriophage lambda. Unfortunately, the term “transfection” is now routinely used to describe the introduction of any DNA or RNA into cells. In this textbook, we use the correct nomenclature: the term “transfection” is restricted to the introduction of viral DNA or RNA into cells with the goal of obtaining virus reproduction.
METHODS
Synthesis of infectious horsepox virus from chemically synthesized DNA
Although smallpox has been eradicated, vaccination against the disease is still carried out in certain populations, e.g., the military. The modern smallpox vaccine, which has some undesirable side effects, shares common ancestry with horsepox virus. However, horsepox virus is extinct, so the necessary experiments to determine if it has a better safety profile could not be done. Might the 212,000-bp horsepox dsDNA genome sequence, available in public databases since 1993, be of use?
To rescue horsepox virus from DNA, ten large DNA fragments from 10 to 30 kb were synthesized by a commercial facility (at a cost of $150,000). The DNAs were transfected into cells that were also infected with a related poxvirus, Shope fibroma virus. The latter is needed to provide proteins necessary for transcription of the viral DNA, which contains promoters that are not recognized by the cellular machinery. The medium from the transfected cells was subjected to plaque assay, and single plaques were shown
