Genetic manipulation of Paramyxoviruses

Paramyxoviruses are enveloped monopartite single stranded negative-sense RNA viruses.

They belong to the Mononegavirales order which is classified in three families;

Paramyxoviridae, Filoviridae, and Bornaviridae. (Rhabdoviridae also belongs to

mononegaviriales), (Samal 2011).

Order MononegaviralesFamily Subfamily Genus Type speciesBornaviridae Bornavirus Borna disease

virusFiloviridae Marburg-like

virusesMarburg virus

Ebola-like viruses Ebola virusParamyxoviridae Paramyxovirinae Avulavirus Newcastle

disease virusHenipavirus Hendra virusMorbillivirus Measles virusRespirovirus Sendai virusRubulavirus Mumps virus

Pneumovirinae Pneumovirus Human respiratory syncytial virus

Metapneumovirus Avian pneumovirus

Paramyxovirus virions are enveloped and can be spherical, filamentous or pleomorphic.

Fusion proteins and attachment proteins appear as spikes on the virion surface. Matrix

proteins inside the envelope stabilise virus structure. The nucleocapsid core is composed of

the genomic RNA, nucleocapsid proteins, phosphoproteins and polymerase proteins.

Genome

5tLack of systems for genetic manipulation of RNP viruses using powerful recombinant DNA technologies has long limited experimental approaches to studying the genetics and biology of negative-strand viruses. Their enormous potential as tools for basic and applied biomedical research stems from the high integrity of RNP genomes within the cell, the mode of gene expression from simply organized genomes, the cytoplasmic replication cycle of most of RNP viruses, and the relatively simple structure of envelopes and holo-virions.

By using recombinant DNA technology, defined virus mutants can be designed to elucidate the basic common principles of RNP gene expression, as well as individual sophisticated features, such as the utilization of overlapping reading frames, RNA editing, RNA splicing, or ambisense gene expression. In addition, viruses are being generated that have never

existed in nature that infect novel target cells, or express foreign proteins. Thus RNP virus research has been aimed at preventing infections with those agents.

The structure and the organization of the genomes of nonsegmented NSV (NNSV) is identical, and is governed by the particular mode of gene expression. The linear, single-strand RNAs of 11-19-kb size represent a succession of individual protein-encoding genes, each defined by an upstream gene start signal and downstream gene end signal. The 3ꞌ and 5ꞌ ends of the RNA are represented by short nontranslated sequences that carry important cis-acting signals for transcription and replication. Transcription starts at the very 3ꞌ end of the genomes producing an approximately 50-nucleotide leader RNA. This is followed by sequential synthesis of the individual mRNAs, giving rise to a gradient of transcripts, steadily decreasing toward the template 5ꞌ end. Thus, the gene order roughly reflects the required amount of products.

After infection of a cell, RNP serves as a template for two RNA synthesis functions, transcription of subgenomic m RNA and replication of full-length RNA. The RNP genomes possess one promoter at the 3i end of the RNA where the virus RNA-dependent RNA polymerase enters for both m RNA transcription and genome replication.

Palese and colleagues described a system that allowed successful generation of biologically active RNPs containing artificial RNA. Transcripts that contained authentic terminal sequences from an influenza virus genome segment and an internal chloramphenicol acetyltransferase (CAT) reporter gene were incapsidated in vitro by purified influenza virus nucleoprotein and the virus polymerase proteins. After transfection of the reconstituted recombinant RNP into influenza virus-infected cells the construct was replicated, transcribed and translated. The helper virus provided the proteins needed for further RNA synthesis and allowed packaging of the synthetic genome segment into progeny virus and passage of CAT activity in tissue culture. Thus, reassortant ('transfectant') viruses that possessed specific alterations in different genome segments could be generated.

Since recombinationRecombination in non-segmented viruses is a very rare event hence large RNAs in the range of 10 to 18 kb have to be manipulated in order to generate an altered virus. In vitro encapsidation is ineffective,probably because of the tighter RNP structure (Baudin et al., 1994). However, experiments indicated that functional encapsidation of preformed RNAs from nonsegmented viruses is possible in principle and also confirmed the location of an RNA encapsidation signal close to the 3' end of the genome.

Krystal and colleagues first succeeded in demonstrating that a short, artificial RNA construct could be rescued inside a cell into a formthat allowed recognition and amplification by the polymerase of a non-segmented virus (Park et al., 1991).The synthetic RNA, which was generated in vitro by T7 RNA polymerase transcription from a linearized plasmid corresponded to a Sendai virus minigenome in which the entire coding region was replaced with the coding region of the CAT reporter gene. This model genome possessed the virus 3'-terminal sequence including the putative promoter for the polymerase and the signal(s) directing leader RNA transcription/release and initiation of mRNA transcription. The 5' end contained the transcription stop/polyadenylation signal derived from the Y-terminal cistron (L) and encoded the antigenomic promoter for replication. In the form of an RNP, this construct thus represents a functional template for replication of RNPs and also, due to the presence of a leader/reinitiation signal, for transcription of positive-stranded

leader RNA and a CAT mRNA. Park et al. (1991) demonstrated that after transfection of the in vitro-transcribed RNA construct into cells, CAT activity was observed after Subsequent infection of cells with Sendai virus.

Furthermore, the artificial RNPs were packaged into infectious virus particles, as demonstrated by successful passage of CAT activity by transfer of cell-free supernatants. Thus, it was confirmed that all cisacting sequences required for encapsidation, initiation ofreplication and transcription of this paramyxovirus reside in the terminal sequences of the genome.Rescue of transfected RNAs by infectious helper virus has also been successful in other paramyxovirus systems such as respiratory syncytial virus, parainfluenza virus type 3 and measles virus. The 5' end appeared to tolerate additional nucleotides. Nucleotides of the 3'-terminal promoter could be replaced by sequences complementary to the 5' end (i.e. the antigenome promoter) A particular feature of the paramyxovirus polymerase, RNA editing, was also found to be template directed (Park and Krystal, 1992)

Practical applications of Paromyxovirus genetic manipulation1. Paramyxovirus vector for transfering foreign gene into skeletal muscle

The objective was to provide Paramyxovirus viral vector that ensures highly efficient transfer of foreign genes into skeletal muscle cells and use thereof. Insulin like growth factor I (IGF-I) plays an important role in the development, maintenance, and regeneration of skeletal. A recombinant Sendai virus (SeV) vector containing LacZ reporter gene and human insulin-like growth factor-I (hIGF-I) gene was used for gene delivery into skeletal muscle. These results demonstrated that Paramyxovirus containing SeV achieves high-level transgene expression in skeletal muscle, and that IGF-I gene transfer using Paramyxovirus vector may have a great potential in the treatment of neuromuscular disorders.

2. Paramyxovirus vector encoding angiogenesis gene and use thereof

Human gene therapy has been clinically applied to therapeutic angiogenesis in order to treat critical ischemic limb. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), an endothelial A cell-specific mitogen, is a potent therapeutic gene for this purpose.

The strategy here is to provide Paramyxovirus vectors encoding angiogenic genes and use thereof. A recombinant Sendai virus (SeV)-mediated gene transfer was used.

Two recombinant SeV vectors were used as therapeutic tools for limb ischemia: one expressing human VEGF165 and the other expressing murine fibroblast growth factor 2 (FGF2). The study revealed that intramuscular administration of recombinant Sendai virus vectors significantly increased transgene expression. Recombinant Sendai virus vectors showed 10- to 100-fold higher expression than plasmid vectors. Moreover, gene therapy on cardiac infarction model animals using a Sendai virus vector expressing FGF2 resulted in an increase in the survival rate compared to individuals to which the control vector was injected. Thus, Paramyxovirus vectors encoding angiogenic genes were confirmed to be effective as gene transfer vectors for ischemic diseases including limb ischemia and myocardiac infarction.

3. Creation of an infectious recombinant Sendai virus expressing the firefly luciferase gene from the 3' proximal first locus

A recombinant non- segmented negative-strand RNA virus, Sendai virus (SeV) of the family Paramyxoviridae, that expresses firefly luciferase was generated. The DNA construct containing the entire open reading frame (ORF) of the luciferase gene followed by the SeV transcription stop and restart signals connected with the conserved intergenic three nucleotides was inserted immediately before the ORF of the viral 3'-proximal nucleocapsid (N) protein gene in a full-length SeV cDNA copy. After intracellular expression of full-length antigenomic transcripts from the engineered cDNA and of the viral n ucleocapsid protein and RNA polymerase from the respective plasmids, a recombinant SeV expressing luciferase activity at a high level was recovered. The inserted luciferase gene was stably maintained after numerous rounds of replication by serial passages in chick embryos. These results indicate the potential utility of SeV as a novel expression vector.

4. Anti-tumor effective Paramyxovirus

The present invention relates to the treatment of tumors by administration of live animal paramyxovirusParamyxovirus from the group APMV3, APMV4, APMV5, APMV6, APMV7, APMV8, APMV9, Mapueravirus and Fer-de-Lance virus are described, which can be used for the production of a medicament for the treatment of tumors. The virus has a selectivity to kill human tumor cells but not human normal differentiated and human normal proliferating cells at the same dose. By genetic engineering the virus can be modified in such a way that one or more genes are added or are replaced by the homologous genes of a related paramyxovirus. By that method the anti-tumor activity of the resulting chimeric virus is enhanced compared to the parental virus.

Genetic Engineering of the Virus (i) The viral genome is sequenced. Based on the sequence, primers for cloning are designed that span unique restriction enzyme recognition sites within the viral genome. By RT-PCR several fragments of the viral genome are cloned as DNA in a plasmid vector like pX8δT. (ii)With the genomic plasmid, mutations, exchange of genes and other types of genetic engineering are carried out on the DNA-level. (iii)By transfection of the genomic plasmid-DNA into cells that express the T7-polymerase together with helper plasmids, recombinant virus can be rescued. Alternatively, cells can be cotransfected with an expression plasmid encoding for T7-polymerase. Additional transgenes are preferably inserted in the intergenic region between M-F or F-HN. The F and HN transgenes may be derived from any APMV, preferably from a virus that has itself strong oncolytic potency. The F and H/HN transgenes may be derived from another paramyxovirus other than APMV. It may be sufficient to express only the heterologous F or the HN protein individually in order to increase the oncolytic potency of the virus.

Chimeric Viruses In order to combine positive features of two different viruses or in order to get rid of negative features it is possible to substitute fragments of the genome with homologous fragments of another virus. That virus may be any related paramyxovirus and is not limited to APMV.

The endogenous genes for the F and HN proteins of APMV can be replaced by heterologous genes from related paramyxoviruses to alter the specificity and tumor-selectivity of the virus.

To allow proper assembly of virions all three proteins M, F and HN can be replaced by the homologous gene segment of a related virus.

In order to decrease the pathogenicity for birds, the gene for the P/V protein is either mutated or replaced by the gene from a related virus. The same procedure can also increase the specificity of the virus for killing of human tumor cells compared to normal cells.

The endogenous L, P and NP proteins can be exchanged. The exchange of these three proteins will have an effect on the replication properties of the virus.

References

Samal, SK (editor) (2011). The Biology of Paramyxoviruses. Caister Academic Press. ISBN 978-1-904455-85-1.