Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 79 (4), 2614-9

Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins

Affiliations

Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins

Takashi Sera. J Virol.

Abstract

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.

Figures

FIG. 1.
FIG. 1.
Inhibition of Rep binding to direct repeats by AZP. (a) Rep binding site in the BSCTV replication origin. The two boxes indicate the direct repeats recognized by the Rep protein. The underlined 19-bp region is the target site of the designed six-finger AZP to block Rep binding. Only the sense strand is shown. (b) Amino acid sequence of the AZP. The underlined amino acids were selected from the nondegenerate recognition code table (17) for recognition of the 19-bp target shown in panel a. The numbers at the top are positions relative to the first amino acid of each recognition helix. (c) Lanes: 1, 32P-labeled probe containing direct repeats; 2, band shift in the presence of 1 nM AZP; 3, band shift in the presence of 1 μM Rep; 4 to 6 and 7 to 9, band shifts in the presence of Rep (1 μM) together with 1 and 10 nM AZP, respectively. In lanes 4 and 7, Rep was added to the binding mixture after incubation of the probe with AZP for 30 min. In lanes 5 and 8, Rep and AZP were mixed together with the probe. In lanes 6 and 9, AZP was added to the binding mixture after incubation of the probe with Rep for 30 min.
FIG. 2.
FIG. 2.
Photographs of WT and AZP-transgenic A. thaliana plants agroinoculated with strain GV3101(pAbar-CFH). (a) Agroinoculated WT (left) and T3 transgenic 1-1A (right) plants. (b) Agroinoculated WT (left) and T3 transgenic 2-1A (right) plants. (c) Magnified image of the gently curling inflorescence of the 2-1A plant indicated by the white rectangular frame in panel b. (d) Magnified image of a typical inflorescence of an agroinoculated WT plant.
FIG. 3.
FIG. 3.
Southern blot analysis of total DNA isolated from agroinoculated WT and T3 transgenic 1-1A and 2-1A plants. (a) DNA bands probed with the DIG-labeled PCR product (200 bp) of the BSCTV genome. Lanes: 1, 50 ng of pCFH digested with EcoRI; 2, 2 μg of total DNA isolated from a whole agroinoculated WT plant; 3, 2 μg of total DNA isolated from a whole agroinoculated 1-1A plant; 4, 2 μg of total DNA isolated from the half of an agroinoculated 2-1A plant that had gently curling inflorescence, indicated by the white frame in Fig. 2b; 5, 2 μg of total DNA isolated from the remaining half of the agroinoculated 2-1A plant, in which no symptoms were observed. (b) Ethidium bromide-stained gel image of total DNA used for the Southern blot shown in panel a. This photograph was taken before processing of the Southern blot. OC, open circular DNA; SC, supercoiled DNA; SS, ssDNA.
FIG. 4.
FIG. 4.
Transgenic plants 4 weeks after agroinoculation. (a) Images of whole plants. (b) Images of rosette leaves. In both panels, a noninfected WT plant, an infected WT plant, a 1-3A plant with minor symptoms, and a 1-3B plant with no symptoms are shown from the left to the right. T3 transgenic plants 1-3A and 1-3B were obtained from T2 line 1-3.

Similar articles

See all similar articles

Cited by 26 PubMed Central articles

See all "Cited by" articles

LinkOut - more resources

Feedback