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.