Basic amino acid mutations in the nuclear localization signal of hibiscus chlorotic ringspot virus p23 inhibit virus long distance movement

Abstract

The p23 is a unique protein in the Hibiscus chlorotic ringspot virus which belongs to Family Tombusviridae Genus Carmovirus. Our previous results showed that the p23 is indispensable for host-specific replication and is localized in the nucleus with a novel nuclear localization signal. To investigate additional function(s) of p23, mutations of basic amino acids lysine (K), arginine (R) and histidine (H) that abolish its nuclear localization, were introduced into a biologically active full-length cDNA clone p223 of HCRSV for testing its effects on virus replication and virus movement in vivo. Primer-specific reverse transcription-PCR was conducted to detect gene transcript level of p23 and viral coat protein separately. Virus replication and its coat protein expression were detected by fluorescent in situ hybridization and Western blot, respectively. The effect of p23 was further confirmed by using artificial microRNA inoculation-mediated silencing. Results showed that the two mutants were able to replicate in protoplasts but unable to move from inoculated leaves to newly emerged leaves. Both the p23 and the CP genes of HCRSV were detected in the newly emerged leaves of infected plants but CP was not detected by Western blot and no symptom was observed on those leaves at 19 days post inoculation. This study demonstrates that when p23 is prevented from entering the nucleus, it results in restriction of virus long distance movement which in turn abrogates symptom expression in the newly emerged leaves. We conclude that the p23 protein of HCRSV is required for virus long distance movement.

Figure 1. Organization of HCRSV genomic RNA and its corresponding open reading frames and predicted proteins (not drawn to scale).

The upper rectangles represent open reading frames. Untranslated region (UTR); putative transcription factor (PTF); RNA-dependent RNA polymerases (RdRps); movement protein (mp); pathogenesis-related gene (prg); coat protein (CP). The dotted vertical line represents a readthrough codon UAG. The 3-dimensional rectangles below represent the corresponding predicted mature proteins. Basic amino acids K, R, and H represent the mutation sites in the nuclear localization signal (NLS) of p23.

Figure 2. Comparative replication of HCRSV wild type (wt) and two p23 mutants.

(A) A single molecule fluorescent in situ hybridization (FISH) method was used to detect virus replication, using a Cy3-labeled cDNA probe (corresponding to the HCRSV p23 coding region at nt 350–301). DAPI stained nuclei (blue-color foci) were superimposed with the differential interference contrast to form a merged image. Kenaf protoplasts were fixed with 4% paraformaldehyde. Cy3 signals were not detected in mock transfected protoplasts. Representative sections for each of the three dimensional images of protoplasts transfected with HCRSV wt and two mutants were shown (mock, wt, H to A and K, R to A, A). Single RNA molecules (red dots) were detected in the protoplasts transfected with in vitro transcript of full-length cDNA clone of HCRSV wt or mutants H to A (H A) or K, R to A, A (K, R A, A ) mutant at 72 hours post transfection. The average density of Cy3 signals in the protoplasts quantified using Velocity 6.1.1 software (PerkinElmer) showed that the replication level was similar for the HCRSV wt and its two mutants. Bar = 2 µm. (B) Comparison of the average Cy3 density measured from 10 protoplasts for mock, HCRSV wt, HCRSV basic amino acid mutants H to A and K,R to A,A, respectively. Standard deviations were shown.

Figure 3. Comparison of HCRSV symptoms on kenaf leaves infected with wild-type (wt) virus and its two mutants at 19 days post inoculation.

HCRSV symptoms were not observed in the mock control. HCRSV wt infected leaf displayed chlorosis. Symptoms were observed in the inoculated leaves (red circles with dotted outline) but not in the newly emerged leaves (white squares with dotted outline) of p223 (H A) and p223 (K, R A, A) mutants. Close-ups of newly emerged leaves and inoculated leaves are shown in inserts with squares and circles, respectively.

Figure 4. Detection of p23 and CP of HCRSV in newly emerged wild type (wt), (H to A) and (K, R to A, A) inoculated kenaf plants at 19 days post inoculation (dpi), respectively.

(A) Both p23 and CP genes were detected in the newly emerged leaves of plants infected with two mutants (H A) and (K, R A, A) at 19 dpi using RT-PCR. Actin gene was used as a loading control. The band intensity of each gene transcript from RT-PCR results was quantified using ImageJ. (B) Detection of HCRSV-CP protein in the wt, mutants H to A and K, R to A, A in the newly emerged kenaf leaves at 19 dpi. The CP expression level was below detection level for the two mutants.

Figure 5. Less severe symptoms were observed in the newly emerged leaves from amiRp23-inoculated kenaf plants which were pre-infected with HCRSV 10 days earlier.

(A) Overlapping PCR to obtain amiRp23 or amiRSO. The a, b, c represent the required fragments to form amiRp23 or amiRSO. PCR fragment d is the product of overlapping PCR from a, b and c. (B) Verification of amiRp23 and amiRSO transient expression in the kenaf leaves using Agrobacterium-infiltration by qRT-PCR. Mock, amiRp23 and amiRSO represent total RNA from mock, amiRp23 and amiRSO Agro-infiltrated kenaf leaves, respectively. Verification of amiRp23 (C) and p23 gene (D) transcript levels in the HCRSV-infected kenaf leaves treated with mock, amiRp23 and amiRSO by qRT-PCR, respectively. V+mock, V+amiRp23 and V+amiRSO represent total RNA from HCRSV infected leaves followed by inoculation with mock, amiRp23 and amiRSO, respectively. (E) Comparison of symptoms (red circles with dotted outline) in the newly emerged leaves between mock (more severe) and amiRp23-inoculated kenaf leaves (less severe) pre-inoculated with HCRSV 10 days earlier, using visual observations. Significant differences of relative amiRp23 and p23 gene between mock with amiRp23 and mock with amiRSO treated plants were calculated using the one sample Student’s t-test. Asterisks (∗∗) indicate significance at 0.01 levels of confidence.