The p23 protein of hibiscus chlorotic ringspot virus is indispensable for host-specific replication

Abstract

Hibiscus chlorotic ringspot virus (HCRSV) possesses a novel open reading frame (ORF) which encodes a putative 23-kDa protein (p23). We report here the in vivo detection of p23 and demonstrate its essential role in viral replication. The expression of p23 could be detected in protein extracts from transfected kenaf (Hibiscus cannabinus L.) protoplasts and in HCRSV-infected leaves. Further, direct immunoblotting of infected kenaf leaves also showed the presence of p23, and transient expression in onion and kenaf cells demonstrated that the protein is distributed throughout the cell. Site-directed mutagenesis showed that mutations introduced into the ORF of p23 abolished viral replication in kenaf protoplasts and plants but not in Chenopodium quinoa L. The loss of function of the p23 mutant M23/S33-1 could be complemented in trans upon the induced expression of p23 from an infiltrated construct bearing the ORF (pCam23). Altogether, these results demonstrate that p23 is a bona fide HCRSV protein that is expressed in vivo and suggest that p23 is indispensable for the host-specific replication of HCRSV. In addition, we show that p23 does not bind nucleic acids in vitro and does not act as a suppressor of posttranscriptional gene silencing in transgenic tobacco carrying a green fluorescent protein.

FIG. 1.

Schematic representation of HCRSV p23 mutants. Mutations were introduced into the full-length cDNA clone p223 (A). The point mutants of p23 were: M23/1T, nucleotide 41 (ATG→ACG), start codon→Thr(B); M23/S33-1, nucleotide 138 (TCG→TAG), amino acid 33 Ser→ stop codon (C); M23/S150-1, nucleotide 489 (TCA→TAA), amino acid 150 Ser→ stop codon (D).

FIG. 2.

Immunoblot detection of p23 in infected kenaf protoplasts and leaves. (A) Total proteins (5 or 10 μg) from infected or mock-inoculated leaves. I represents HCRSV-infected leaves. M represents mock-inoculated leaves. The purified p23 protein expressed from E. coli was used as a positive control. (B) Equal loading of total proteins (5 μg) isolated from protoplasts inoculated with tp223 at 0, 12, 24, and 36 h p.i.

FIG. 3.

Whole-leaf immunoblot detection of p23. Fixed, cleared leaves were subjected to immunohistochemical detection. Alternate substrates were used for the detection of p23 (blue) and coat protein (red). Mock-inoculated control leaves (A) show only background coloration, whereas HCRSV-infected leaves (B) show intense punctate blue staining or intense red staining to indicate the presence of p23 or coat protein, respectively.

FIG. 4.

Northern blot analyses of kenaf protoplasts and plants inoculated with in vitro transcripts of the p23 mutants. (A) Kenaf leaves at 7 days p.i. with tp223, tM23/1T, tM23/S33-1, and tM23/S150-1, respectively. (B) Transfected protoplasts at 24 h p.i. The 18S RNA bands (at the bottom) indicate equal RNA loadings.

FIG. 5.

Complementation between the p23 mutant M23/S33-1 and a rescue construct expressing p23, pCam23. tM23/S33-1 was used to inoculate kenaf plants after infiltration with pCam23. Total RNA was extracted at 2, 7, and 14 days p.i. and subjected to Northern blot and RT-PCR analyses. (A) Northern blot with a DIG-labeled cRNA probe specific to the 3′ 0.8 kb of the HCRSV sequence. Genomic RNA was detected in leaves infiltrated with pCam23 but not with p1300. (B) RT-PCR analysis with p23 specific primers detected a 630-bp band in leaves infiltrated with pCam23 but not with p1300. M, DNA marker.

FIG. 6.

Infectivity of HCRSV p23 mutants in C. quinoa. (A) One percent agarose gel electrophoresis of RT-PCR products amplified from total RNA (isolated from local lesions appearing in C. quinoa) with p23 specific primers. (B) Northern blot of p23 mutants in protoplasts. Total RNA was extracted at 24 h p.i. The 18S RNA bands indicate similar RNA loading.

FIG. 7.

Northern blot showing GFP accumulation in the infiltrated leaves of the N. benthamiana GFP transgenic plants. Total RNAs were extracted from the leaves 3 and 6 days after infiltration with pBin35S-mGFP5 (pBin; lanes 2 and 5), pBin35S-mGFP5/pCam23 (p23; lanes 3 and 6), pBin-35S-mGFP5/T2b (T2b; lanes 4 and 7), or noninfiltrated leaves (Ni; lane 1), respectively, and monitored by Northern blot analysis. The blot was hybridized with digoxigenin-labeled GFP full-length antisense RNA probes. 18S RNA indicates equal RNA loading.