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By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa calafornica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPVpcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) is the type species of the Baculoviridae, a large family of insect viruses, and has a circular, supercoiled DNA genome of -134 kb (1, 2). It has been extensively exploited for the overexpression of eukaryotic genes and is being engineered for possible use as a viral insecticide. Despite its widespread use, little is known about the mechanism by which AcMNPV DNA replicates. Eight regions distributed around the genome have been identified as putative origins of DNA replication (3-6). Seven of these origins (ori) are located within homologous regions (hr) (Fig. 1), which contain repeats of closely related imperfect palindromes (8). One origin is located within the HindIII-K fragment, which does not contain a hr (9).To date only a putative helicase gene (10) and a putative DNA polymerase gene (11,12) have been identified as essential for baculovirus DNA replication. In addition, a gene (pcna) encoding a protein resembling proliferating-cell nuclear antigen (PCNA), which is a DNA polymerase processivity factor in other systems, has been identified in Ac-MNPV (13), but its role in DNA replication has not been determined.With a transient replication assay, six large regions of the AcMNPV genome were identified that contain one or more genes involved in DNA replication (7). In this report, this assay was used for the identification of six genes encoding proteins essential for AcMNPV DNA replication and three genes whose products stimulate DNA replication.MATERIALS AND METHODS Cells and Virus. Spodoptera frugiperda Sf9 cells (14) were cultured in TNM-FH medium (15), supplemented with 10% fetal bovine serum (FBS). The E2 strain ofAcMNPV (16) was used as wild-type virus. Routine cell culture maintenance and virus infection procedures were carried out as described (17).Plasmid Constructs. The nine replication genes were identified within six regions previously shown to be essential for DNA replication (ref. 7; see also Fig. 1). Subclones of each region were tested for their ability to substitute for the larger parental clone. The following clones were constructed. lef-) is located on the EcoRI-O fragment (18) and was cloned as an Nru I-EcoRI fragment (m.u. 7.5-8.7, ref. 2) into plasmid pUC19. lef-2 is on EcoRI-I (19, 20) and was cloned as an Mlu I fragment (m.u. 1.9-2.6) with Mlu I-Bgl II linkers into the BamHI site of pUC19. The DNA polym...

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the Bunyaviridae. Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS S gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS S -expressing leaf sectors suggests that the tospoviral NS S protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.RNA silencing involves a sequence-specific degradation which is induced by overabundant RNA and by doublestranded RNA (dsRNA) molecules and which can target transgenes as well as homologous endogenous genes. RNA silencing was first described for plants (35,50) and over recent years has been described for other organisms, where it is also referred to as cosuppression, posttranscriptional gene silencing (17), or RNA-mediated virus resistance (3,11,30) in plants, quelling in fungi (9), or RNAi in animals (19). Building blocks of the gene-silencing pathway proved to have remarkable similarities in the different organisms and hence suggest an ancient role of gene silencing in pathogen resistance or development (10,25,53). One of the key intermediary elements in the RNA silencing pathway is dsRNA, which is recognized by a dsRNAspecific nuclease (5) to yield small (21 to 23 nucleotides) short interfering RNAs (siRNAs) (21). These siRNAs subsequently serve as guides for cleavage of homologous RNA molecules. In plants, versions of transgenes that produce dsRNA molecules have been shown to be very potent activators of RNA silencing (47). As all RNA viruses replicate through formation of dsRNA intermediates, these are potential targets of the RNA silencing mechanism. Indeed, antiviral RNA silencing has been shown to occur in nature and has been proposed as a natural defense mechanism protecting plants against viruses, resulting in resistance (1, 43).To counteract the RNA silencing mechanism of their host, plant viruses have developed ways to evade or neutralize this response. Over recent years, RNA silencing-inhibiting proteins have been identified in several plant viruses. Among the best-studied examples are the helper component-proteinase (HC-Pro) of the potyvirus Potato virus Y (PVY) and the 2b protein of Cucumber mosaic virus (CMV) (7, 40). Other plus-strand RNA (and some DNA) viruses also have been found to suppress gene silencing, and ...

The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference tests. In addition, more offspring were produced on the virus-infected pepper plants, and this effect also was found for TSWV-infected Datura stramonium. Thrips behavior was minimally influenced by TSWV-infection of host plants with only a slight preference for feeding on infected plants. Offspring development was positively affected since larvae hatched earlier from eggs and subsequently pupated faster on TSWV-infected plants. These results show a mutualistic relationship between F. occidentalis and TSWV.

White spot syndrome virus (WSSV), the sole member of the virus family Nimaviridae, is a large double-stranded DNA virus that infects shrimp and other crustaceans. By alignment of three completely sequenced isolates originating from Taiwan (WSSV-TW), China (WSSV-CN) and Thailand (WSSV-TH), the variable loci in the genome were mapped. The variation suggests the spread of WSSV from a common ancestor originating from either side of the Taiwan Strait to Thailand, but support for this hypothesis through analysis of geographical intermediates is sought. RFLP analysis of eight Vietnamese WSSV isolates, of which six were collected along the central coast (VN-central) and two along the south coast (VN-south), showed apparent sequence variation in the variable loci identified previously. These loci were characterized in detail by PCR amplification, cloning and sequencing. Relative to WSSV-TW, all VN-central isolates showed a~8?5 kb deletion in the major variable region ORF23/24, whereas the VN-south isolates contain a deletion of~11?5 or~12?2 kb, compared to a~1?2 or~13?2 kb deletion in WSSV-CN and WSSV-TH, respectively. The minor variable region ORF14/15 showed deletions of various sizes compared with WSSV-TH for all eight VN isolates. The data suggest that the VN isolates and WSSV-TH have a common lineage, which branched off from WSSV-TW and WSSV-CN early on, and that WSSV entered Vietnam by multiple introductions. A model is presented for the spread of WSSV from either side of the Taiwan Strait into Vietnam based on the gradually increasing deletions of both 'variable regions'. The number and order of repeat units within ORF75 and ORF125 appeared to be suitable markers to study regional spread of WSSV.

White spot syndrome virus (WSSV), member of a new virus family called Nimaviridae, is a major scourge in worldwide shrimp cultivation. Geographical isolates of WSSV identified so far are very similar in morphology and proteome, and show little difference in restriction fragment length polymorphism (RFLP) pattern. We have mapped the genomic differences between three completely sequenced WSSV isolates, originating from Thailand (WSSV-TH), China (WSSV-CN) and Taiwan (WSSV-TW). Alignment of the genomic sequences of these geographical isolates revealed an overall nucleotide identity of 99.32%. The major difference among the three isolates is a deletion of approximately 13 kb (WSSV-TH) and 1 kb (WSSV-CN), present in the same genomic region, relative to WSSV-TW. A second difference involves a genetically variable region of about 750 bp. All other variations >2 bp between the three isolates are located in repeat regions along the genome. Except for the homologous regions ( hr1, hr3, hr8 and hr9), these variable repeat regions are almost exclusively located in ORFs, of which the genomic repeat regions in ORF75, ORF94 and ORF125 can be used for PCR based classification of WSSV isolates in epidemiological studies. Furthermore, the comparison identified highly invariable genomic loci, which may be used for reliable monitoring of WSSV infections and for shrimp health certification.

The complete nucleotide sequence of the S RNA of tomato spotted wilt virus (TSWV) was determined. The RNA is 2916 nucleotides long and has an ambisense coding strategy. The sequence contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted Mr of 52"4K and one in the viral complementary sense which encodes the viral nucleocapsid protein of Mr 28"8K. Both proteins are expressed by translation of two subgenomic RNA species that possibly terminate at a long stable hairpin structure, located at the intergenic region. The structure of this RNA segment resembles that of the arthropod-borne phleboviruses (family Bunyaviridae). The absence of significant sequence homology between TSWV and bunyaviruses infecting animals suggests that TSWV should be considered as a representative of a new genus within the Bunyaviridae.