Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts and plants.

The replication of wheat dwarf virus (WDV) in protoplasts derived from a Triticum monococcum suspension cell system was investigated. The production of circular viral double-stranded DNA (dsDNA) forms consistent with the replication of the viral genome was observed. In comparison to whole plants, the production of viral single-stranded DNA (ssDNA) was reduced, possibly due to only low levels of viral coat protein being produced in the protoplasts. Mutations introduced into the viral coat protein open reading frame (ORF) did not affect the ability of the viral DNA to replicate, and a deletion of ca. 400 bp was tolerated. However, these mutations abolished the infectivity of the viral genome when agroinoculated onto wheat plants, providing evidence that, contrary to the case for the bipartite geminiviruses, the coat protein is essential for infection by WDV.     

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.     

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.     

Identification of the initiation sequence for viral-strand DNA synthesis of wheat dwarf virus.

The intergenic region of the circular single-stranded DNA genome of geminiviruses contains a sequence potentially able to fold into a stem-loop structure. This sequence has been reported to be involved in viral replication by serving as the origin for rolling-circle replication. However, in wheat dwarf virus (WDV) a deletion of 128 bp, removing this sequence, surprisingly does not prevent de novo viral DNA synthesis, but instead abrogates the processing of replicative intermediates into monomeric genomes. This deletion mutant permitted us to study the initiation of viral-strand DNA synthesis independently from its termination and also to identify the sequence within which rolling-circle DNA replication of WDV begins. We have mapped the initiation site of replication to a pentanucleotide, TACCC, a sequence that occurs twice in the large intergenic region of WDV: it is found in the right half of the stem-loop sequence and again 170 bases upstream where it is part of a 15 nucleotide sequence highly homologous to the right half of the stem-loop sequence. Here we show that viral-strand DNA synthesis efficiently initiates at both sequences.     

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation ( Ds ), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep⁺) and non-replicating (rep⁻) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep⁺) or mutant, inactive derivatives of these genes (rep⁻). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds -vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 10⁵-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.     

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.     

Agrobacterium-mediated viral vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture

A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Rep-supplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.     

Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.     

GRAB proteins,novel members of the NAC domain family,isolated by their interaction with a geminivirus protein

Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.     

Methods for construction of adenovirus vectors

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.     

Viral Vectors for Gene Delivery and Expression in the CNS

Viral vectors have emerged as an important tool for manipulating gene expression in the adult mammalian brain. The adult brain is composed largely of nondividing cells, and therefore DNA viruses have become the vehicle of choice for neurobiologists interested in somatic gene transfer. Recombinant viral vectors based upon adenovirus or herpes simplex virus have been created in which a gene essential for viral replication is removed and a gene of interest is inserted in the viral genome. While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral gene expression within target cells. The defective HSV vector has been used to transfer genes into the adult rat brain. This vector has also been used for analysis of the preproenkephalin promoterin vivo,and important regions of this promoter have been identified using this technique. A modification ofin situPCR has been developed as an adjunctive tool for sensitively documenting the presence of vector DNA within target cells duringin vivopromoter studies. Finally, the adenoassociated virus vector has been used as the first fully defective DNA viral vector, which also eliminates any contamination by helper viruses. This vector can transfer genes into the mammalian brain and has shown significant behavioral recovery in a rodent model of Parkinson's disease. Future work will undoubtedly result in still more diverse and improved vectors; however, these studies have documented the importance of viral vectors to both basic neurobiology and the potential treatment of neurologic disease.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.