The term ‘reverse genetics systems’ for negative sense RNA viruses refers to systems of introducing mutations into the viral genome and determining the resulting phenotype. In order to be able to manipulate the RNA genome of RNA viruses, it is first necessary to ‘convert’ it into a DNA copy that produces the RNA genome in the presence of a DNA dependent-RNA polymerase. There are three reverse genetics systems- the helper virus system, the plasmid rescue system and the full-length rescue system (Theriault et al., 2004).
The full-length rescue system aims to produce virus entirely from cDNA in vitro. The requirements for the system are a DNA genome plasmid, a DNA dependent RNA polymerase, the viral nucleocapsid and polymerase proteins and any additional helper proteins (figure 1.6). This system has been developed for RSV and hMPV (Bukreyev et al., 1997; Herfst et al., 2004). Firstly a method for creating the RNA genome from the
cDNA clone was required. By cloning the genome under the control of a T7 DNA dependent RNA polymerase promoter, transcription resulted in a copy of the antigenome RNA being produced in the cytoplasm of the cell. The T7 DNA dependent RNA
M2-1 VVT7 Genome plasmid T7 Pro N P L Assay of infectious virus T7 RNA pol T7 RNA pol
Figure 1.6 Model of the negative sense RNA virus full length rescue system. The components required to create a reverse genetics system for a negative sense RNA viruses are show here. VVT7 (A) is an engineered vaccinia virus used to constitutively express T7 DNA dependent RNA polymerase (T7 RNA pol) in infected HEp2 cells (B). These cells are then transfected with a genome plasmid (C) under the control of a T7 polymerase promoter (T7 Pro) that produces an RNA copy of the antigenome upon transcription by the T7 RNA polymerase, which is then terminated using a hepatitis ribozyme. Simultaneously, the cells are transfected with expression plasmids for the helper proteins (D). For RSV, these are the N, P, L and M2-1 proteins. These cells then have all the components
necessary to form RNP complexes, transcribe and replicate the genome (E). Transcription and replication of the cloned RNA genome result in the production of infectious virus which can be assayed (F).
A C B D E F 41
dependent RNA polymerase (Fuerst et al., 1986) or by using BSR-T7 cells that have been engineered to express T7 DNA dependent RNA polymerase (Buchholz et al., 1999). With the T7 polymerase present in the cells, expression plasmids for the N, P and L proteins (under the control of a T7 promoter) were transfected into cells to provide the helper proteins in the cytoplasm of the cell. In this way, the anti-genome RNA, and the N, P, L and M2-1 proteins are able to form a functional unit and the synthetic genome is
replicated and transcribed. By using the T7 polymerase, these RNA transcribed in the cytoplasm as is seen in natural pneumovirus infection, rather than in the nucleus. When the entire virus genome is used, infectious virus is produced and can be assayed. This system has the advantage of producing infectious virus that can be assayed in vitro
and in vivo. The effect of mutations to the viral genome can be assessed in terms of viral
fitness, RNA production, and tissue tropism. However, pneumovirus genomes are 13 to 16kb and cDNAs are technically difficult to create and manipulate. Hence historically before the creation of a full-length rescue system, a minigenome was used to determine the minimal requirements for viral replication and transcription. This minigenome was used to establish helper virus and plasmid rescue systems, as described below. In the plasmid rescue system, a simpler synthetic ‘minigenome’ (figure 1.7) is used in place of the full-length genome clone. A minigenome has a reporter gene inserted between the viral replication and transcription signals and the reporter protein expression can be used as a measure of RNP complex function. This is termed a plasmid rescue system (figure 1.8) and has advantages and disadvantages compared to the full-length rescue system. In the plasmid rescue system no virus is produced, whereas full-length system produces infectious virus. However, the plasmid rescue system allows
manipulation of not only the cis-acting non-translated genome sequences but also of the helper proteins.
The helper virus rescue system uses infectious virus to provide the trans-acting proteins to replicate and transcribe the minigenome in vitro (figure 1.9). The difference between
Figure 1.7 Schematic of the APV minigenome and dicistronic minigenome
constructs. For the minigenome (A), the gene end (GE) signal (5’ UAGUUAAUU 3’, mRNA sense), gene start (GS) signal (5’ GGGACAAGU 3’, mRNA sense), leader (Le) sequence and trailer (Tr) sequence were cloned flanking the CAT (chloramphenical acetyl transferase) reporter gene under the control of the T7 RNA polymerase promoter (T7) in the pt7.2 plasmid. The RNA fragment resulting from transcription by the T7 RNA polymerase and the hepatitis delta virus ribozyme sequence (δ)creates the exact viral Le sequence by self cleaving (indicated with the blue arrow). For the dicistronic minigenome (B), an addition reporter gene was added, a luciferase gene. The APV minigenomes were provided by Dr. J. Smith (Marriott et al., 2001).
T7 GS GE Le CAT Tr δ Pt7.2 plasmid 43 T7 CAT GS Le Tr δ Pt7.2 plasmid GE GS Luciferase GE
A
B
M2-1 VVT7 Minigenome plasmid T7 Pro N P L Assay of reporter protein T7 RNA pol T7 RNA pol
Figure 1.8 Model of the negative sense RNA virus plasmid rescue system. The
components required to create a reverse genetics system for a negative sense RNA viruses are show here. VVT7 (A) is an engineered vaccinia virus used to constitutively express T7 DNA dependent RNA polymerase (T7 RNA pol) in infected HEp2 (B) cells. These cells are then transfected with a minigenome plasmid (C) under the control of a T7 polymerase promoter (T7 Pro) that produces an RNA copy of the anti-minigenome upon transcription by the T7 RNA polymerase, which is then terminated using a hepatitis ribozyme.
Simultaneously, the cells are transfected with expression plasmids for the helper proteins (D). For RSV, these are the N, P, L and M2-1 proteins. These cells then have all the components necessary to form RNP complexes, transcribe and replicate the genome (E). Transcription of the minigenome produces reporter mRNA which, after translated by the cell, produces reporter protein (F).
A C B D E F
APV