Viral Infections

Plants

Poultry

Mammals

Humans

Productivity

Best way to improve the productivity is to control the viral infections

Dealing with the Deadly…..

• Base analogs

• Inactivation of the viral receptors, enzymes

• Interrupting the post translational modifications of proteins

In many cases the viruses remained recalcitrant to the human intelligence just by simple mutations in their genome leading to variant enzymes

Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins

Takashi Sera

JOURNAL OF VIROLOGY, Feb. 2005, p. 2614–2619

Torrey Mesa Research Institute, San Diego, California

Zinc Finger MotifCharacteristics of the Zinc finger motif family:

Function: The DNA-binding motif is found as part of transcription regulatory proteins.  Binding: Fingers bind to 3 base-pair subsites and specific contacts are mediated by

amino acids in positions : -1, 2, 3 and 6 relative to the start of the alpha-helix. Contacts mainly involve one strand of the DNA. Where proteins contain multiple fingers, each finger binds to adjacent subsites within a larger DNA recognition site thus allowing a relatively simple motif to specifically bind to a wide range of DNA sequences.

 

Cys-(X)2-Cys-(X)12-His-(X)3-His.

1st Base 2nd Base 3rd Base

G Arg His Arg

A Gln Asn Gln

T Thr Ser Thr

C Glu Asp Glu

Position 6 Position 3 Position -1

Artificial Zinc Finger Protein

Rep binding site in the BSCTV replication origin

19bp region recognized by the AZP in the BCTV origin

Beet severe Curly Top Virus origin

Beet severe Curly Top Virus

• BSCTV belongs to Curtovirus group of Geminivirus

• It is characterized by monopartite circular ssDNA genome

• Infects a wide variety of dicots

• Symptoms range from curling and stunting of inflorescences, deformation of floral structures, leaf curling and deformation, vein swelling, accumulation of anthocyanins and finally death

• Replication is intiated by binding of Rep on the direct repeats in the origin

Artificial zinc finger Protein

Lane 1: Labeled probe containing direct repeats; Lane 2: Band shift in the presence of 1 nM AZP; Lane 3: Band shift in the presence of 1M Rep Lanes 4,7: Band shifts in the presence of 1M Rep with 1nM AZ P(Rep was added to the binding mixture after incubation of the probe with AZP for 30 min) Lanes 5,8: Band shifts in the presence of Rep (1M) together with 1nM AZP respectively (Rep and AZP

were added to the binding mixture at the same time). Lanes 6,9: Band shifts in the presence of Rep (1M) together with 1nM AZP respectively (AZP was added to the binding mixture after incubation of the probe with Rep for 30 min)

AZP inhibits the binding of Rep to the direct repeats in BCTV origin

AZP Vs Rep

Photographs of WT and AZPtransgenic 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 typicalinflorescence of an agroinoculated WT plant.

Transgenic AZP A.thaliana

AZP Transgenics are resistant to plants against BCTV infection

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 BSCTVgenome. Lane 1: 50 ng of pCFH digested with EcoRI;Lane 2: 2 g of total DNA isolated from a whole agroinoculated WT

plant; Lane 3: 2 g of total DNA isolated from a whole agroinoculated

1-1A plant; Lane 4: 2 g of total DNA isolated from the half of an agroinoculated 2-1A plant that had gently curling inflorescence;Lane 5: 2 g 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

Southern Blotting

AZP successfully inhibits the replication of BSCTV

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.

a Each value is the number of WT or T3 transgenic plants showing each symptom after agroinoculation with BSCTV. b The line numbers correspond to the T2 lines. T3 transgenic plants used for the agroinoculation were obtained from these T2 lines.c Same phenotypes as healthy, noninfected WT Col ecotype plants.d Same phenotypes as healthy, noninfected WT Col ecotype plants, except for gentle curling of a couple of tops of inflorescences.e Plants were as tall as WT plants, but all tops of inflorescences were curled or deformed. Some stems in some plants were thicker than WT stems. No symptom were observed on rosette leaves.f Observed symptoms included curling and stunting of all inflorescences, deformation of floral structures, short and thick stems, leaf curling and deformation, vein swelling, and accumulation of anthocyanins.

• One or more AZPs can be employed to tackle more than one variants of the viruses• This strategy can be analysed in tackling mammalian viruses also