Virus-mediated gene transfer

1. Virus-mediated gene transfer

2. Virus-mediated gene transfer • Viral genome doesn’t integrate into plant genome. • Viral vectors are episomal vectors, therefore they have high copy number per cell and they are not subjected to the “position effect”. The gene product is very rapidly accumulated. • Viral genome sequences are excellent source of promoters, enhancers and other components useful for designing gene vectors. • Virus is systemically spread in the plant body. • Generally have wide host range.

3. Most notables • CaMV based vectors. • Gemini virus based vectors. • TMV based vectors. • Basic features: • Broad host range, virulence, easy mechanical transmission and seed transmission. • Carry additional genetic information within the packaging limit. Viruses that are rod shaped or have multipartite genome or contain a helper or satellite component offer potential for carrying extra genetic material.

4. CaMV

5. CaMV Circular (top) and linearized (bottom) genome maps of Cauliflower mosaic virus (CaMV). ORFs or ORF segments encoding a protein are represented by boxes shaded in different patterns according to the different putative functions of the genes (vertical lines for the putative movement protein, slanted lines for the capsid protein and horizontal lines for the reverse transcriptase protein). The symbols used are the followings: § movement protein active site, * RNA binding site, ? protease active site, ¨ reverse transcriptase active site, D RNAse H consensus sequence. The map starts at the intergenic region of the circular genome for convenience, the arrow shows the position of the promoter and the number 1 indicates the origin of DNA replication.

6. 19S RNA VII I VI II 8031 bp III V VI 35S RNA (polycistronic) CaMV • dsDNA virus. Packaged as nucleosome. • Mechanical and aphid mediated transmission • Virion DNA alone or cloned CaMV DNA is infectious when simply rubbed on leaves • Up to 106 copies per cell. 3-4 weeks for systemic infection through plant. Gene I: plasmadesmata movement Gene IV: translation transactivation Gene V: reverse transcriptase Gene III/IV: assembly Gene II/VI: inclusion bodies

7. Gene I uncoating nucleus Inclusion body (gene VI) Gene III/IV transcription assembly Gene V Reverse transcription 35S RNA 19S RNA Gene IV translation CaMV activity in plant cell

8. Expression of a bacterial gene in plants by using a viral vector Several properties of the cauliflower mosaic virus (CaMV) indicate that it could provide a useful vector for gene transfer in higher plants: (1) it has a relatively small double-stranded genome that can be easily manipulated in vitro; (2) cloned viral DNA is infectious when rubbed onto healthy leaves; (3) virus spreads throughout the plant and can be found in most cells at high copy number. Two regions of the CaMV genome—open reading frames (ORFs) II and VII—do not seem to be essential for infection, as both can be either deleted or expanded by small inserts of foreign DNA. No functional genes have yet been introduced into these ORFs. Here we report the replacement of CaMV ORF II by the R67 plasmid-encoded dihydrofolatereductase (DHFR) gene; this gene (dhfr) confers resistance to methotrexate in Escherichia coli. The chimaeric viral DNA can be stably propagated in turnip plants and the dhfr gene is expressed, producing a functional enzyme. Nature 310, 511 - 514 (09 August 1984)

9. Mammalian Metallothionein Functions in Plants A recombinant cauliflower mosaic virus (Ca-MTII) was constructed by inserting a cDNA clone of Chinese hamster metallothionein II into the ORF II of the cloned virus pCa-BB1. Systemically-infected Brassica campestris tissue contained metallothionein at a level of 0.5% of the soluble leaf protein. This efficient expression conferred 4X the Cd-binding capacity when compared with Ca-BB1 infected leaves. Ca-MTII-infected leaves exposed to 1 mM CdCl2 bound all the free Cd whereas uninfected leaves possessed 43.8 nmol free Cd per milligram of protein. This may be responsible for Cd resistance in the Ca-MTII plant cells. Metallothionein is the first mammalian gene product shown to be functional in plants. Bio/Technology5, 1053 - 1056 (1987)

10. Expression of Mammalian Metallothionein Suppresses Glucosinolate Synthesis in Brassica campestris Transfection of Brassica campestris leaves with cauliflower mosaic virus (CaMV) harboring a mammalian metallothionein (MT) cDNA at the ORFII position lowered the glucosinolate (GS) concentration to approximately one-half the level in leaves infected with wild-type CaMV. This suppression was independent of the plant's sulfate status, suggesting that the pathways for protein (MT) and GS biosynthesis were competing for S on an equal basis. The expression of MT may have lowered the endogenous levels of Cys, 3'-phosphoadenosine-5'-phosphosulfate, or Met, all of which are required for GS synthesis in B. campestris. These results indicate that the introduction of structural genes coding for high levels of specific amino acids can be used to alter the production of non-proteinaceous molecules within plants. Plant Physiol. (1990) 93, 522-524

11. Challenges with CaMV vector Small insertions (10-30 bp) in various sites abolished infectivity. Only gene II could tolerate insertion of significant size and could be entirely removed But the largest insert tolerated so far is 256-531 bp. Complicated polycistronic design (ATG of cloned DNA must not interfere with the termination of gene I). CaMV genome was inserted into Ti vector to integrate into genome. Due to these limitations, CaMV vectors have not be widely used.

12. Gemini viruses

13. V1 C1 WDV V2 C2 AC1 TGMV DNA B TGMV DNA A BV1 BV1 AC3 AC2 Gemini viruses: smallest viral genome ~2.7 kb WDV, TGV and MSV: ssDNA genome replicates as dsDNA intermediate V2: capsid protein V1: systemic spread C1 and C2: replication Monopartite Bipartite AC1: replication AV1: Coat protein AC2: transactivation of sense genes AC3: delayed or attenuated symptoms BC1 and BV1: spread AV1

14. V1 C1 MCS (multi-cloning site) C2 Gemini viruses dsDNA form of virus genome is infectious and coat protein gene is not required for systemic infection Most have insect mediated transmission. No mechanical transmission. Overcome by cloning viral genome into Ti plasmid and carrying out “Agroinfection” WDV genome allows insertion of up to 3 kb foreign sequence pWDV2 V2 deletion vector

15. Gene amplification and expression in plants by a replicating geminivirus vector The use of plant DNA viruses as vectors for the transfer of foreign genes to plants offers two potential advantages over other, existing methods of producing transgenic plants. First, these viruses systemically infect whole plants, thus obviating the need for the difficult and time-consuming step of regeneration from transformed single cells or protoplasts. Second, the viruses replicate as separate, autonomous entities within the plant's cells so that any gene cloned in a plant DNA-virus vector would be amplified to high copy number, a feature that differs from methods that produce transgenic plants by the chromosomal integration of foreign DNA. To date, attention has focused on the development of vectors based on the cauliflower mosaic virus (CaMV), but their use is hampered by the narrow range of plants infected by CaMV, and by practical limitations on inserting foreign DNA that are imposed by the biology of CaMV. Here we describe the use of vectors based on the gemini tomato golden mosaic virus (TGMV) to introduce the neomycin phosphotransferase (neo gene) into plants. Our results indicate that geminivirus-derived vectors should be useful not only for amplification of gene expression by the systemic infection of plants, but also for heritable gene amplification by the integration of stable master copies of the vector into the plant chromosomal DNA. Nature334, 179 - 182 (14 July 1988)

16. RNA virus for foreign gene expression The vast majority of plants viruses consist of plus strand RNA. Engineering gene expression vectors based on RNA viruses has awaited the successful cloning of the viral genome into in vitro transcription vectors from which infectious transcripts can be obtained. cDNA cloning of Brome Mosaic Virus (1984) and Tobacco Mosaic Virus (1986) were reported. In these cases CP gene was replaced with cat gene and the resulting vectors were expressed in barley or tobacco protoplasts.

17. TMV ssRNA virus of 6395 bases with 4 ORFs 5’ cap 30 kDa CP tRNA like 126 / 183 kDa 126 / 183 kDa: replicase complex translated from a single frame (183 kDa is generated by readthrough of a leaky amber termination codon of 130 kDa gene 30 kDa: movement protein 17.5 kDa: capsid protein

18. Insert (size limit) TB2 30 kDa CP 126 / 183 kDa 3’ TMV based vectors Foreign gene is inserted into 3’ end of MP in such a way that native CP promoter drives the expression of foreign gene, and a related virus (ORSV: Odontoglossom ring spot virus) CP promoter drives the expression of native CP ORF

19. TMV based vectors

20. Joshi RL, Joshi V, Ow DW. (1990) EMBO J. 9:2663-9. BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells. Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs alpha, beta and gamma that have been cloned into in vitro transcription vectors from which infectious transcripts can be obtained. The BSMV genome has been engineered here to serve as an expression vector in plant protoplasts. Open reading frame (ORF) b of RNA beta, encoding a non-structural protein, was replaced by the firefly luciferase (luc) reporter gene to yield RNA beta 2-luc. In the presence of both RNAs alpha and gamma, RNA beta 2-luc mediated efficient expression of the luc gene upon transfection into tobacco and maize protoplasts. This expression ranged from 20- to 123-fold higher than the luciferase activity obtained from transfection with a luc gene mRNA. Replication of RNA beta and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern analysis, indicating that the high level of luc gene expression using RNA beta 2-luc resulted from RNA amplification. ORFa of RNA beta, encoding the coat protein, was also replaced by the luc gene to yield RNA beta 1-luc. Although transfection of RNA beta 1-luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further increase of luciferase activity was observed in the presence of RNAs alpha and gamma, or RNAs alpha, beta and gamma, suggesting that the deleted sequences within ORFa are cis-acting for replication of RNA beta. Our results demonstrate that a foreign gene can be expressed by replacement of a viral non-structural protein gene that is essential for virus multiplication in plants, leading to a potential strategy for virus 'containment' with use of 'disarmed' plant viral vectors.

21. Joshi RL, Joshi V, Ow DW. (1990) EMBO J. 9:2663 RNA α ORFb encodes a non-structural protein that is essential for virus multiplication in whole plant. ORFb ORFc ORFa Organization of BSMV genome RNA β ORFd RNA γ BSMV based vectors ORFb ORFc RNA β 1 ORFd ORFc ORFa RNA β 2 ORFd ORFb ORFc RNA β1-luc luc ORFd

22. http://www.lsbc.com/ GENEWARE® is a unique and extremely versatile technology, used by LSBC to test the function of novel genes and proteins they encode (gene prototyping), and to manufacture complex soluble proteins in bulk. GENEWARE® is one of three types of viral vectors within LSBC’s viral vector technology platform. This form of transient, plant viral-vector-based gene expression utilizes a modified Tobacco Mosaic Virus (TMV), or similar RNA viral replicon, carrying its normal housekeeping genes plus an introduced new RNA promoter and structural gene to encode a new protein of commercial interest. LSBC’s dual subgenomic promoter system is designed for the coordinated expression of a wide range of soluble (free) protein products with high efficiency. GENEWARE's basic principle is to use a safe vector modified from a virus to place any gene (or a large number of genes) within a test organism. The organism then manufactures the gene's protein product, which can be collected and purified efficiently in LSBC's pilot and commercial bioprocessing facilities. Kilogram amounts of a wide array of complex biopharmaceuticals can be produced rapidly with low capital investment relative to conventional biotechnology manufacturing operations.

24. Coat Protein Fusion and Virus-Like Particle Vectors LSBC’s second type of viral vector technology for gene expression and protein manufacturing converts plant viruses, such as Tobacco Mosaic Virus (TMV) and relatives, into a carrier and display vehicle for peptides. By inserting the gene sequence(s) for small proteins (peptides) onto selected regions of TMV’s coat protein (CP) gene, the virus can be made to display the new peptide on the CP surface upon assembly. The CP promoter is very strong and thus the yield of peptides can be very high. Because upon assembly each TMV virion particle consists of over 2,100 CP subunits, the resulting product is a virus displaying over 2,100 copies of a protein product per particle. The peptides on the viral CP can be fused through a labile amino acid bridge, so the peptide product can be recovered and separated from the rest of the virus post translationally. More often, the viral particle acts as a carrier or display vehicle for peptides of interest. This technology finds major utility in the field of vaccine development, in which case TMV becomes a viral antigen display vehicle. The semicrystalline array of a very high number of repeating antigens can stimulate the immune system to respond with potent cellular and humoral immunity to these vaccines. TMV rod-shaped particles can also be coated with peptides and proteins chemically or biochemically, to achieve the same type of effects as described with the genetic CP fusion approach. Because more than one sequence of peptide can be displayed on the viral surface, this type of vector enables the development of multivalent vaccines, which are useful in the prevention and control of complex current and emerging diseases. LSBC and its collaborators have successfully constructed several candidate vaccines using this CP/VLP vector technology. Representative examples of such products include a feline parvovirus (panleukopenia) vaccine, a bovine foot and mouth disease vaccine, and an experimental human papillomavirus vaccine.

26. Hybrid TMV Vectors for Transient RNA Vaccination LSBC’s third type of viral vector technology for gene expression consists of a hybrid viral replicon that integrates several separate features of viral architecture and virus-host biology. Because TMV and related plus-strand RNA viruses are members of the Alphavirus superfamily, consisting of RNA replicons, LSBC has designed a composite system that is manufacturable in commercial quantities cost effectively and has a limited capacity to effect gene expression in mammalian tissue. However, these vectors cannot integrate into the mammalian chromosome and thus do not persist in the body. These vectors, still under development, could form the core for a new type of transient RNA vaccination. Instead of producing protein, which becomes the product, these new vectors would encode protein in situ only after injection as a vaccine. These hybrid viral vectors are constructed by growing TMV or related viruses in Nicotiana plants, purifying them to homogeneity, and then isolating their coat protein (CP) subunits away from their native RNA genome. The CP subunits can be native, or carry on them fused surface antigens-essentially as LSBC’s CP/VLP vectors do. But the hybrid system then coalesces the CP subunits around a plus strand of non-native, synthetic RNA containing a mammalian Alphavirus replicating element. This element is important because native TMV RNA cannot replicate in mammalian tissue. The TMV virion structure now consists of a repetitive antigen—either monovalent or multivalent—carried and displayed to the immune system on the CP, plus the information to replicate and translate new protein transiently in mammalian antigen-presenting cells, such as Dendritic cells in the skin. While this system is earlier in development relative to LSBC’s other viral vector technologies, initial feasibility studies in vivo using model antigens have been highly encouraging and suggest that this hybrid virus technology could form the basis of a new multivalent, bi-functional (gene/protein) vaccine strategy to combat cancers and many infectious diseases.

28. McCormick et al. PNAS Vol. 96, Issue 2, 703-708, January 19, 1999 Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants Abstract Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 µg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

29. Schematic diagram of the viral expression vector used to produce murine 38C13 scFv NHL antigen in Nicotiana benthamiana host plants. Shown are the sequences encoding rice α -amylase signal peptide fused in-frame to the SphI site (underlined), introduced by PCR, 5' of the 38C13 heavy chain. Also indicated in the linear map are the positions of the SP6 transcription start site, the 126-kDa, 183-kDa, and 30-kDa proteins from TMV, and the tomato mosaic virus coat protein, as well as pBR322 sequences for bacterial propagation.

30. Protein analysis of N. benthamiana plants 2 weeks after inoculation. Lanes 1 and 2, Coomassie-stained SDS/PAGE of IF extract [secreted proteins were isolated by vacuum infiltration, centrifugation, and sterile filtration (IF extracts)] from viral constructs expressing rice α -amylase protein (lane 1) or of 38C13 scFv IF extract (lane 2) run under nonreducing conditions. Lanes 3 and 4, Western blot of a duplicate gel probed with anti-38C13-specific monoclonal antibody S1C5 containing the control extract (lane 3) or 38C13 scFv (lane 4). Lanes 5 and 6 show S1C5 Western blots of 38C13 scFv-containing IF prepared under reducing conditions (but separated by PAGE under nonreducing conditions) or IF containing an 38C13 scFv protein that has no cysteine at amino acid 3. Lanes 7 and 8, Coomassie stain of the starting IF extract (lane 7) and of single-pass, S1C5 affinity-purified 38C13 scFv protein (lane 8). Lane 9, silver stain of affinity-purified protein.