PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions 2564 (box WK 1 ) and 2859 (box WK 2 ). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK 1 box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoterTb-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35STNtWRKY12 and PR-1aTGUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

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C ircularization of the genome of RNA viruses plays an essential role in the replication and translation of viral RNAs. Formation of panhandle structures by base pairing between complementary sequences at the 5Ј and 3Јtermini of several genera of minus-strand RNA viruses has been known already for some time (1). Recently, it became clear that cyclization by RNA-RNA interactions may also play an essential role in the replication of positive-strand RNA viruses such as f laviviruses (2, 3). However, for a growing number of positive-strand RNA viruses, circularization seems to be mediated by protein bridges between the 5Ј and 3Ј termini of the viral RNA. On entry into the host cell, the genome of positive-strand RNA viruses has to serve as messenger for the translation of viral proteins including replication factors. A number of viral RNAs resemble cellular mRNAs by having a 5Ј-cap structure (m 7 GpppN) and 3Ј-poly(A) tail. In cellular mRNAs, these structures permit the formation of a protein bridge by interactions between eIF4G and eIF4E from the cap-binding complex of translation initiation factors and the poly(A) binding protein (PABP) associated with the poly(A) tail (4, 5). Picornaviruses do not have a cap structure, but the internal ribosome entry site in the 5Ј untranslated region (UTR) of the RNA binds initiation factors and a poly(rC) binding protein that mediate the interaction with PABP. Circularization of Poliovirus RNA seems to play a role both in translation and replication of the viral RNA (6, 7).The genomic RNAs of many viruses lack a poly(A) tail, and elements in the 5Ј and͞or 3Ј UTR are believed to compensate for the absence of a poly(A) tail (8-12). Recently, the rotavirus (family Reoviridae) NSP3 protein was shown to interact with eIF-4G and with the 3Ј UTR of the nonpolyadenylated viral mRNAs. Both interactions were required for efficient translation of the viral mRNA, and NSP3 was suggested to mimic the function of PABP (13). Here, we report evidence that the coat protein (CP) of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) also may act as a functional equivalent of PABP.RNAs 1 and 2 of the tripartite AMV genome encode the replicase proteins P1 and P2, respectively. P1 contains methyltransferase-and helicase-like motifs, whereas P2 contains polymerase-like motifs. RNA 3 encodes the movement protein and CP, which is translated from a subgenomic messenger, RNA 4. The RNAs contain a 5Ј-cap structure but the 3Ј ends are not polyadenylated (14). At their 3Ј termini, RNAs 1-4 contain a homologous sequence of 145 nucleotides that can be folded into two alternative conformations: a linear array of hairpins with flanking AUGC sequences that represents a strong CP binding site or a tRNA-like structure that represents the promoter for minus-strand RNA synthesis (15). A mixture of the three genomic RNAs becomes infectious only after addition of CP or its messenger, RNA 4, to the inoculum (16). Initiation of infection requires binding of a few molecules of CP to the 3Ј termini of the g...

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3-untranslated regions (UTRs) of both RNAs deleted (R1⌬/2⌬ construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1⌬/2⌬ construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1⌬/2 (3-UTR of RNA 1 deleted), R1/2⌬ (3-UTR of RNA 2 deleted), or R1⌬/2⌬ plus R3. This demonstrates that the 3-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3-termini of AMV RNAs is not required for assembly of virus particles.RNA-dependent RNA polymerase (RdRp) proteins of many plus-strand RNA viruses have been expressed in heterologous systems, including Escherichia coli (15, 23-25, 31, 35, 42, 61), Saccharomyces cerevisiae (17, 36), insect cells (4,14,30,43,63), and mammalian cells (26). The availability of in vitro active RdRp has greatly facilitated research into the mechanisms of plus-strand virus replication. Mutant RNA polymerases can be expressed despite their inability to support viral replication. In this way, insight can be gained into the roles of the various domains of viral replicase proteins.Alfalfa mosaic virus (AMV) (for reviews, see references 6 and 18) is a tripartite plus-strand RNA virus belonging to the family Bromoviridae (Fig. 1A). The AMV replication complex consists of two viral proteins, P1 and P2, and possibly host proteins. P1, encoded by RNA 1, has homologies to known methyltransferase domains in its N-terminal half and homologies to helicase domains in its C-terminal half (12, 21). P2, encoded by RNA 2, contains the RNA polymerase domain characterized by the GDD motif (for a review, see reference 33). AMV RNA 3 codes for the viral movement protein (P3) and the coat protein (CP), which is translated from a subgenomic mRNA 4. All three genomic RNAs of AMV contain 5Ј-and 3Ј-untranslated regions (UTRs), which are highly structured and presumed to contain important cis-acting regulatory elements for viral replication and translation (32,52,54,55,58,59).The 3Ј-terminal 145 bases...

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