1 Department of Virology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
2 Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA
3 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Correspondence
Amit Gal-On
amitg{at}volcani.agri.gov.il
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ABSTRACT |
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Present address: Department of Plant Pathology, Iowa State University, Ames, IA 50011-1020, USA.
These authors contributed equally to this work.
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INTRODUCTION |
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Mixed infections involving Cucumber mosaic virus (CMV) and a number of other viruses have been found to result in viral synergy in some plant species (Fukumoto et al., 2003; Palukaitis & Kaplan, 1997
; Pio-Ribeiro et al., 1978
; Poolpol & Inouye, 1986
; Sano & Kojima, 1989
; Wang et al., 2002
), whilst CMV interferes with resistance to some potyviruses (Murphy & Kyle, 1995
; Sáenz et al., 2002
) and vice versa (Choi et al., 2002
; Wang et al., 2004
). The 2b protein encoded by CMV enhanced pathogenicity in Nicotiana benthamiana when expressed from a Potato virus X (PVX) vector (Brigneti et al., 1998
; Lucy et al., 2000
), but did not facilitate the systemic infection of Plum pox potyvirus (PPV) when expressed from PPV in the same host (Sáenz et al., 2002
). Thus, the 2b protein may not be the sole CMV determinant for facilitation of interviral synergy involving CMV.
The absence of the CMV 2b protein was shown not to affect virus replication in protoplasts (Ding et al., 1996; Soards et al., 2002
), but did affect the extent and pattern of CMV movement in tobacco (Soards et al., 2002
) and cucumber (Ding et al., 1995a
). Insertion of an early translational termination sequence or complete deletion of the 2b gene had the same effect on the accumulation of CMV in protoplasts and whole plants (Ding et al., 1996
), as well as on the ability of a chimaeric CMVumbravirus to promote cell-to-cell movement of the phloem-limited Potato leafroll virus (Ryabov et al., 2001
). The 2b protein was shown to be localized primarily in the nucleus (Lucy et al., 2000
; Mayers et al., 2000
). A nuclear localization signal (NLS) of CMV Q strain was identified and localization to the nucleus was determined to be necessary, but not sufficient, for silencing suppression in N. benthamiana, whereas neither the ability to suppress silencing nor nuclear localization alone appeared to be sufficient for enhancement of the pathogenicity of PVX (Lucy et al., 2000
).
The effect of the 2b protein on synergy between CMV and potyviruses has not been examined. Moreover, interactions between CMV and potyviruses in cucurbit hosts showed several characteristics that are different from interviral interactions in N. benthamiana or Nicotiana tabacum (Choi et al., 2002; Pruss et al., 1997
; Sáenz et al., 2002
; Wang et al., 2002
, 2004
). Thus, we examined the role of the CMV 2b protein compared with the 3a movement protein (MP) and capsid protein (CP) in synergy between CMV and the potyvirus Zucchini yellow mosaic virus (ZYMV) in several cucurbit species. In addition, we assessed the role of nuclear localization of the 2b protein in enhancing pathogenicity in cucurbit hosts.
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METHODS |
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Inoculation procedure and virus detection.
Cotyledons of cucurbit seedlings were inoculated by bombardment with cDNA clones of ZYMV-AT, ZYMV-AGII or ZYMV-AGII carrying foreign genes, as described by Gal-On et al. (1997). Fny-CMV and Fny-CMV
2b RNA transcripts were synthesized individually from the appropriate cDNA templates, as described previously (Soards et al., 2002
; Zhang et al., 1994
). The mixed in vitro CMV RNA transcripts were bombarded with a suspension of tungsten particles in calcium nitrate buffer at pH 8·5 (Gal-On et al., 1997
) to inoculate N. glutinosa leaves and squash cotyledons. In double inoculations, ZYMV-AGII cDNA and Fny-CMV
2b transcripts were bombarded separately onto the same cotyledons. CMV RNA accumulation was detected by RT-PCR. In addition, RT-PCR of viral progeny derived from ZYMV-AGII or ZYMV-AGII carrying the CMV 2b gene or various mutated 2b genes was performed by using a one-tube, single-step RT-PCR method, as described by Arazi et al. (2001)
, with the primers described below. ZYMV CP accumulation was detected by Western blot analysis, as described previously (Wang et al., 2002
).
Construction of CMV MP, CP and 2b genes for expression via the ZYMV-AGII vector.
The Fny-CMV MP, CP and 2b genes were amplified by PCR and cloned into the vector pGEM-T (Promega) using the following primers: for the MP gene, 5'-GATGCTGCAGTCAGCTTTCCAAGGTACCAGTAGG-3' and 5'-CTGACTGCAGCATCACAGTGTCAAGACCGTTAACCACCTGCGG-3' (PstI sites underlined); for the CP gene, 5'-GATGCTGCAGTCAATGGACAAATCTGAATCAACCAGT-3' and 5'-TGACTGCAGCATTACAGTGTCGACTGGGAGCACTCCAGA-3' (PstI sites underlined); and for the 2b gene, 5'-AAACTGCAGAGCGAATTGAACGTAGGTGCAATG-3' and 5'-ACGGACGTCGACGAAAGCACCTTCCGCCCATTC-3' (PstI and SalI sites underlined). The MP and CP genes were cloned into the ZYMV-AGII (AGII) viral expression vector by using the PstI sites. The 2b gene was introduced into the PstI/SalI sites, as described by Arazi et al. (2001). The resultant clones were designated AGII-MP, AGII-CP and AGII-2b, respectively, and were used for standard bombardment inoculation as described above.
Mutagenesis of the putative NLSs of the Fny-CMV 2b gene.
Mutagenesis of the 2b NLSs was done according to the method of Kunkel et al. (1987), using the following primers: 5'-GCTCGTATGGTGGAGGCTAGCCACAAACAGAATCGA-3' for 2b
NLS1; 5'-CGTATGGTGGAGGCTGCGGCAGCAGCAGCGAGCCACAAACAGAATCGA-3' for 2b6AlaNLS1; 5'-TCTCACAAACAGAATGGTCACAAAAGTCCCAGCGAGAGA-3' for 2b
NLS2; and 5'-TCTCACAAACAGAATGCTGCTGCTGCTCACAAAAGTCCCAGCGACAGA-3' for 2b4AlaNLS2. 2b
NLS1+2 was constructed by mutagenesis of the plasmid containing 2b
NLS1, using the primer for mutagenesis of 2b
NLS2. The pGEM-2b clone served as a template for single-stranded DNA generation and mutagenesis with the appropriate primers. The resultant clones were designated pGEM-2b
NLS1 (deletion of the first putative NLS1), pGEM-2b6AlaNLS1 (replacement of the putative NLS1 residues with six alanine residues), pGEM-2b
NLS2 (deletion of the second putative NLS2), pGEM-2b4AlaNLS2 (replacement of the putative NLS2 residues with four alanine residues) and pGEM-2b
NLS1+2 (deletion of both NLS1 and -2). The modified 2b genes were inserted into the ZYMV-AGII vector by using the PstI and SalI sites, as for the cloning of the native 2b gene described above (Arazi et al., 2001
). The resultant clones, designated AGII-2b
NLS1, AGII-2b6AlaNLS1, AGII-2b
NLS2, AGII-2b4AlaNLS2 and AGII-2b
NLS1+2, were used for standard bombardment inoculations as described above.
Verification of gene stability in progeny viruses.
RT-PCR of the recombinant viruses harbouring the 2b gene or its mutants was performed with primers flanking the cloning region on the AGII genome: 5'-AGCTCCATACATAGCTGAGACA-3' (forward primer from the NIb gene) and 5'-TGGTTGAACCAAGAGGCGAA-3' (reverse primer from the CP gene), as described by Arazi et al. (2001). To confirm the authenticity of the ZYMV-AGII vector in plants infected with ZYMV-AGII expressing 2b or its mutated variants, RT-PCR was performed on the HC-Pro sequence of ZYMV-AGII compared with ZYMV-AT, as described by Gal-On (2000)
, using the following primers: 5'-GTGTTCGAGGTAGAGACGACG-3' (forward) and 5'-CAGCTGCTCCTCGAGTTTAATG-3' (reverse).
Nuclear localization assay using the yeast nuclear import system.
The nuclear import assay of the 2b protein and its mutants was performed by using the pNIA vector according to Rhee et al. (2000). Native and mutant 2b genes were fused to mLexA-Gal4AD fusion in the pNIA vector by using the BamHI/PstI sites in the primers 5'-BamHI-2b (5'-CGGGATCCGTGAATTGAACGTAGGTGCAATG-3'; BamHI site underlined) and 3'-PstI-2b (5'-GCTGCAGGAAAGCACCTTCCGCCCATTCGTT-3'; PstI site underlined), to obtain clones pNIA-2b, pNIA-2b
NLS1, pNIA-2b6AlaNLS1, pNIA-2b
NLS2 and pNIA-2b
NLS1+2. The Agrobacterium tumefaciens genes virD2 and virE2 were used as positive and negative controls (Tzfira & Citovsky, 2001
), respectively, for nuclear localization in the pNIA expression vector (Rhee et al., 2000
). The yeast L40 strain was transformed and the nuclear import assay was performed according to Rhee et al. (2000)
.
Nuclear localization assay in onion cells.
The intact 2b gene and the mutant variants 2bNLS1, 2b
NLS2, 2b4AlaNLS2 and 2b
NLS1+2 were amplified by PCR and cloned in place of the VIP1 gene in the pRTL2-GUS-VIP1 vector (Tzfira et al., 2001
), resulting in an N-terminal fusion of 2b to the
-glucuronidase (GUS) reporter protein. Nuclear localization of fusion proteins GUS2b, GUS2b
NLS1, GUS2b
NLS2, GUS2b4AlaNLS2 and GUS2b
NLS1+2 was examined by following the method of Varagona et al. (1992)
. The fusion constructs were bombarded (Varagona et al., 1992
) onto the inner face of onion inner epidermal layers, covering approximately 3050 cells for each bombardment, by using a Helios portable gene gun at a pressure of 150 p.s.i. Onion inner epidermal layers were peeled and placed on Petri dishes containing 6 % agar and 100 µg ampicillin ml1. The bombarded onion epidermal layers were assayed histochemically with X-glucuronide for GUS expression. After blue staining was visible, tissues were stained immediately with the nuclear-specific stain 4,6-diamidino-2-phenylindole (DAPI) 16 h after bombardment, as described by Varagona et al. (1992)
. Photomicrographs were prepared by using differential-interference optics.
Yeast two-hybrid proteinprotein interaction assay.
The native 2b and the 2b mutant genes were amplified by PCR as BamHIPstI fragments, using the primers 5'-BamHI-2b and 3'-PstI-2b and cloned into the corresponding sites of pSTT91 (TRP1+) (Sutton et al., 2001), producing fusions with the LexA gene. These clones were designated pSTT91-2b, pSTT91-2b
NLS1, pSTT91-2b6AlaNLS1, pSTT91-2b
NLS2, pSTT91-2b4AlaNLS2 and pSTT91-2b
NLS1+2. The gene encoding A. thaliana karyopherin
protein (AtKAP
) was cloned into pGAD424 (LEU2+; Clontech), producing a fusion with the GAL4 activation domain, as described previously (Ballas & Citovsky, 1997
). For the two-hybrid assay, the potential interactors were introduced into the Saccharomyces cerevisiae strain TAT7 (L40-ura3) (Hollenberg et al., 1995
) and grown for 2 days at 30 °C on a leucine-, tryptophan- and histidine-deficient medium. Histidine prototrophy indicated proteinprotein interaction (SenGupta et al., 1996
). Qualitative determination of
-galactosidase activity was performed as described by Stachel et al. (1985)
. High-fidelity Pfu DNA polymerase (Stratagene) was used in all PCRs and all DNA constructs were verified by dideoxynucleotide sequencing.
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RESULTS |
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The second approach to determine whether the Fny-CMV 2b protein was involved in pathogenicity in cucurbits did not depend on movement by CMV. In this approach, the 2b gene was expressed from the viral expression vector ZYMV-AGII (Arazi et al., 2001). This approach also took advantage of the symptom attenuation by the ZYMV-AGII vector to evaluate the potential roles of other CMV genes in pathogenicity in cucurbits. The CMV genes for MP, CP and 2b were cloned between the NIb- and CP-encoding sequences of the ZYMV-AGII expression vector (Fig. 2
a). The inserted genes were designed to create in-frame translational fusions with the ZYMV-AGII-encoded polyprotein. Proteolysis of the nascent AGII polyprotein by the NIa protease in trans was predicted to yield expressed CMV-encoded proteins with one additional amino acid residue (serine) at the N-terminus and seven amino acid residues (VDTVMLQ) at the C-terminus. All of the modified viruses (designated AGII-MP, AGII-CP and AGII-2b) were inoculated by bombardment into cucurbit hosts at the cotyledon stage and the symptoms caused by infection with the modified ZYMV-AGII were examined in cucumber, melon and squash plants. The modified viruses AGII-MP, AGII-CP and AGII-2b were detectable by ELISA by 68 days post-inoculation, similar to infection by the parental virus ZYMV-AGII. Our efforts to express the full-length 1a and 2a genes via the ZYMV-AGII vector were unsuccessful, probably because of a cloning-size limitation of the ZYMV-AGII vector.
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Pathogenicity of the CMV 2b protein and the effect of the putative NLSs
We examined whether the pathogenicity associated with the Fny-CMV 2b protein in cucurbits required an NLS. A putative NLS 22KKQRRR27 (NLS1) (Fig. 3) was deleted from the Fny-CMV 2b protein to produce 2b
NLS1 and the mutant 2b
NLS1 was expressed from ZYMV-AGII (AGII-2b
NLS1). The expression of 2b
NLS1 from AGII-2b
NLS1 was not able to enhance disease symptoms, compared with the parental ZYMV-AGII in infected squash (Fig. 2e
), melon or cucumber (data not shown). In addition, the ZYMV-AGII CP accumulated to a similar level in squash plants that were infected with AGII, AGII-2b or AGII-2b
NLS1 (Fig. 2g
). The stability of the sequences encoding 2b and 2b
NLS1 within the AGII expression vector was confirmed by sequencing the RT-PCR products that were amplified from viral RNA extracted from squash plants infected by AGII-2b and AGII-2b
NLS1 (Fig. 2h
).
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A second putative NLS motif (NLS2), 33RRER36 (Fig. 3), was predicted in the Fny-CMV 2b protein by using the PredictNLS server (http://cubic.bioc.columbia.edu/services/predictNLS/). This sequence was completely conserved in all subgroup I CMV strains and varied by only one amino acid (E35 to A35) in subgroup II CMV strains (Fig. 3
). There were only five amino acid residues between the putative NLS1 and NLS2 sequences. To examine the role of the putative NLS2 in 2b-mediated pathogenicity, the NLS2 was either deleted (2b
NLS2) or substituted by four alanine residues (2b4AlaNLS2). Infection of cucurbit hosts with ZYMV-AGII expressing either 2b
NLS2 or 2b4AlaNLS2 reduced or eliminated the pathogenicity caused by the parental AGII-2b in squash and cucumber (data not shown).
Nuclear import of the CMV 2b protein and its NLS mutants
Nuclear localization of the 2b protein and its mutants was examined by two different methods (Fig. 5). In the first method, histochemical analysis was performed on onion epidermal cells following particle bombardment with a construct allowing transient expression of genes fused to the GUS reporter gene (Varagona et al., 1992
). Specifically, the intact 2b gene and its NLS-modified mutants were fused to sequences encoding the C-terminus of the GUS reporter gene in the expression vector pRTL2-GUS. Following bombardment, GUS expressed from the control construct diffused through the cytoplasm and there was no apparent accumulation of the GUS gene product in the nucleus (Fig. 5a
). By contrast, expression of GUS fused to the 2b gene product was localized in the nucleus (Fig. 5a
), as was the positive control fusion between the nuclear protein VIP1 (Tzfira et al., 2001
) and the GUS reporter gene. Interestingly, the GUS protein fused to 2b
NLS1 (GUS2b
NLS1) was localized in the onion nucleus as effectively as the GUS2b fusion protein (Fig. 5a
). These results indicated that deletion of NLS1 did not affect nuclear localization of the 2b protein. Moreover, GUS gene product fused to 2b
NLS2 (GUS2b
NLS2) or to 2b4AlaNLS2 (GUS4AlaNLS2) was also localized in the nucleus (Fig. 5a
). These results suggested that either NLS1 or NLS2 is sufficient to localize the 2b protein in the nucleus.
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A second method was used to confirm that the native 2b protein localized to the nucleus, by fusion of the wild-type 2b protein and its mutants to the mLexAGal4AD fusion in the pNIA system (Rhee et al., 2000). The mLexAGal4AD chimaeric protein is unable to localize to the nucleus, as the NLS in mLexA is disabled. Due to this disability, the transcriptional activator Gal4AD cannot activate the mLexA operon in yeast cells, resulting in arrest of yeast-cell growth on histidine-deficient medium (Rhee et al., 2000
). Only when the test protein fused to the mLexAGal4AD chimaera possesses a functional NLS will mLexA be able to target the mLexA operator into yeast nuclei and allow yeast-cell growth on media without histidine (Rhee et al., 2000
; Tzfira et al., 2001
). Yeast cells were transformed with pNIA expressing 2b or 2b with modified NLSs. All the transformed cells grew well on non-selective medium (Fig. 5b
). On selective medium, however, cells transformed with pNIA expressing a fused wild-type 2b grew as well as those transformed with fusions containing the A. tumefaciens VirD2 (Fig. 5b
), corroborating that 2b is a nuclear protein. Removal of either NLS domain (NLS1 or NLS2) did not affect 2b nuclear localization, as 2b
NLS1- or 2b
NLS2-transformed yeast cells were able to grow on selective medium (Fig. 5b
). Moreover, substitution of NLS1 or NLS2 with alanine residues (2b6AlaNLS1 or 2b4AlaNLS2, respectively) did not affect nuclear transport (Fig. 5b
and data not shown). However, removal of both NLS domains (2b
NLS1+2) completely abolished 2b protein-mediated nuclear transport, as did expression of a fusion to A. tumefaciens VirE2 (Fig. 5b
).
Interaction of NLS1 and NLS2 with nuclear localization protein AtKAP
It is known that protein localization to the nucleus requires the co-operation of a karyopherin-like molecule for the docking process with the nuclear membrane pore (Suntharalingam & Wente, 2003) and that plant-pathogen proteins use the host nuclear import machinery for their nuclear import (e.g. Ballas & Citovsky, 1997
; Tzfira et al., 2002
). Therefore, we examined whether a plant RNA virus-encoded nuclear protein, such as the 2b protein of CMV, used a similar mechanism.
We first tested whether the native 2b protein interacted with AtKAP (Ballas & Citovsky, 1997
) in the yeast two-hybrid system (SenGupta et al., 1996
). Our results showed that the native 2b protein bound to AtKAP
, as determined by an enzymic assay for the expression of
-galactosidase (Fig. 6
). The 2b/AtKAP
interaction activity was scored as 100 %. Deletion of both NLS domains (2b
NLS1+2) disrupted the interaction between 2b and AtKAP
completely, as no
-galactosidase activity was observed (Fig. 6
). On the other hand, deletion or alanine substitution of either NLS1 or NLS2 did not seem to affect the interaction between 2b and AtKAP
(Fig. 6
).
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DISCUSSION |
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It was demonstrated previously that deletion of the 2b gene from RNA 2 of Fny-CMV caused slower viral movement, lower virus accumulation and amelioration of disease symptoms in tobacco, but did not affect virus replication (Soards et al., 2002). In this study in squash, Fny-CMV
2b exhibited movement limited from the inoculated cotyledons to only the first true leaf. Similarly, either deletion of the 2b gene from RNA 2 of the Q strain of CMV, a member of CMV subgroup II, or introduction of a translation terminator after the third amino acid resulted in the inability of the virus to infect cucumber plants systemically, but the virus could still infect N. glutinosa systemically (Ding et al., 1995a
, b
), with a delay and milder symptoms. The 2b protein was not required for the replication of either CMV or its satellite RNA (Ding et al., 1995b
), but was shown to play a role in promoting cell-to-cell movement (Shi et al., 2003
) and long-distance movement (Ding et al., 1995a
; Ji & Ding, 2001
; Soards et al., 2002
). Whether this is due to a direct effect on movement or an effect on the suppressor activity of the CMV 2b protein that affects movement indirectly cannot be determined, although differences in pathogenicity mediated by different 2b proteins did not correlate with either increased RNA accumulation or more rapid movement per se (Shi et al., 2002
). There are differences in plasmodesmata network connection, size and density between veins and the companion cell of solanaceous and cucurbit plants (van Bel, 1993
). Thus, it is conceivable that the 2b protein is more important for CMV long-distance movement in squash than in tobacco. It is also possible that the CMV 2b protein is less effective in suppression of host responses in squash than in Nicotiana species.
In contrast to symptomatic infection by ZYMV-AGII-2b of the CMV-tolerant cucumber cv. Delila, we have shown previously that mixed infection of the same host with ZYMV-AG and Fny-CMV was symptomless (Wang et al., 2004). This discrepancy in disease symptom elicitation between ZYMV-AGII-2b infection and a mixed infection by ZYMV-AG+Fny-CMV may be due to differences in the relative levels of expression of the 2b genes in each cell in two distinct systems, as well as an additive suppressor function to host RNA silencing. In addition, in mixed infection, the proportion of the infected cells containing both HC-Pro and 2b might be much lower than with the viral vector, where every infected cell contained both the HC-Pro and 2b proteins.
ZYMV-AGII is an asymptomatic, attenuated ZYMV strain derived from the severe strain ZYMV-AT, in which the sequence of the FRNK motif was altered to FINK (Gal-On, 2000). Restoration of disease symptoms by expression of Fny-CMV 2b protein via ZYMV-AGII suggests that the 2b protein may have substituted for the disabled HC-Pro in interactions with host factors, eliciting disease symptoms. Perhaps the 2b protein substituted for the disabled HC-Pro in interactions with host microRNAs, as potyviral-related disease symptoms could, in part, be accounted for by interference with targeting and/or cleavage functions of host microRNAs by HC-Pro (Kasschau et al., 2003
). Thus, examination of interactions between ZYMV-AGII HC-Pro or Fny-CMV 2b and cucurbit microRNAs may provide specific insights into disease symptom restoration by the 2b protein.
We have demonstrated that the 2b protein of a subgroup IA strain of CMV localized to the host nucleus, has two NLSs and that either of the NLSs is sufficient to localize the 2b protein into the nucleus (Figs 5 and 7). Interestingly, both of the NLS sequences were found to be associated with symptom elicitation in cucurbits (Figs 2, 4 and 7
). Moreover, we have provided evidence that both NLSs are capable of binding to AtKAP
, an A. thaliana karyopherin that is known to be an essential nuclear membrane protein (Figs 6 and 7
).
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It has been shown that plant viral proteins, as with other nuclear proteins, need to dock on the outside of the nuclear pore complex in order to enter the nucleus (Görlich & Mattaj, 1996). This has been shown previously only for structural proteins of DNA viruses, such as Cauliflower mosaic virus (Leclerc et al., 1999
) and Tomato yellow leaf curl virus (Kunik et al., 1998
) and a negative-strand RNA virus, Sonchus yellow net virus (Goodin et al., 2001
). Docking may occur via karyopherin
proteins, analogous to the importin
proteins of animals (Görlich & Mattaj, 1996
), as was observed for the A. tumefaciens VirD2 protein, but not the VirE2 protein (Ballas & Citovsky, 1997
). In this study, we showed an interaction between an A. thaliana karyopherin and a non-structural protein of an RNA virus that possesses two NLSs. This indicates that either of the two NLSs of the Fny-CMV 2b protein is capable of interaction with the importin-like docking and nuclear transport system. Each NLS of the viral protein bound separately to the karyopherin, although deletion of both prevented binding. Therefore, modification of either NLS of Fny-CMV 2b protein did not prevent 2b nuclear transport through the nuclear pore complex, but did affect intranuclear activity, preventing symptom elicitation. Whether this occurs by preventing 2b interactions with microRNAs needs to be established.
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ACKNOWLEDGEMENTS |
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Received 3 May 2004;
accepted 30 June 2004.