Principles of Virology. Jane Flint

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).

BOX 3.8

      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.

BOX 3.9

      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 to contain horsepox virus, as determined by viral genome sequencing. The rescued horsepox virus protected immunized mice against a lethal challenge with vaccinia virus.

      This work is the first complete synthesis of a poxvirus using synthetic biology methodology. Some argued that the work enabled the rescue of smallpox virus. However, these concerns are spurious, as no new methods were developed by this work. The infectivity of DNA copies of viral genomes had been known for many years when this work was undertaken.

       Noyce RS, Lederman S, Evans DH. 2018. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One 13:e0188453.

      (ii) (–) strand RNA viruses. Genomic RNA of (–) strand RNA viruses is not infectious, because it can be neither translated nor copied into (+) strand RNA by host cell RNA polymerases (Chapter 6). Two different experimental approaches have been used to develop infectious DNA clones of these viral genomes (Fig. 3.12B and C).

      The recovery of influenza virus from cloned DNA is achieved using an expression system in which cloned DNA copies of the eight RNA segments of the viral genome are inserted between two cellular promoters, so that complementary RNA strands can be synthesized (Fig. 3.12B). When all eight plasmids carrying DNA for each viral RNA segment are introduced into cells, infectious influenza virus is produced.

      When the full-length (–) strand RNA of viruses with a nonsegmented genome, such as vesicular stomatitis virus (a rhabdovirus), is introduced into cells containing plasmids that produce viral proteins required for production of mRNA, no infectious virus is recovered. Lack of infectivity is thought to be a consequence of the hybridization of fulllength (–) strand RNA with (+) strand mRNAs produced from plasmids encoding viral proteins. Such hybridization might interfere with association of the (–) strand RNA with the N protein, which is required for copying by the viral RNA-dependent RNA polymerase. In contrast, when a fulllength (+) strand RNA is transfected into cells that have been engineered to synthesize the vesicular stomatitis virus nucleocapsid protein, phosphoprotein, and polymerase, the (+) strand RNA is copied into (–) strand RNAs. These RNAs initiate an infectious cycle, leading to the production of new virus particles.

      dsRNA viruses. Genomic RNA of dsRNA viruses is not infectious because ribosomes cannot access the (+) strand in the duplex. The recovery of reovirus from cloned DNA is achieved by an expression system in which cloned DNA copies of the 10 RNA segments of the viral genome are inserted under the control of a promoter for bacteriophage T7 RNA polymerase (Fig. 3.12D). When all 10 plasmids carrying DNA for each viral dsRNA segment are introduced into cells, infectious reovirus is produced.

       Types of Mutation

Recombinant DNA techniques allow the introduction of many kinds of mutation at any desired site in cloned DNA (Box 3.10). Deletion mutations can be used to remove an entire gene to assess its role in reproduction, to produce truncated gene products, or to assess