Cloning of Large Positive‐Strand RNA Viruses

Abstract

Full‐length, biologically active cDNA clones of the positive‐strand RNA plant viruses are indispensable for investigating the functions of viral genes and control elements as well as generating virus‐derived gene expression and silencing vectors. Even though engineering of such clones for 4‐ to 10‐kb viral RNAs has become routine, it remains a challenging task for 15‐ to 20‐kb RNA genomes of the monopartite viruses in a family Closteroviridae. This unit describes strategic considerations and techniques used to generate an infectious cDNA clone of a closterovirus. The use of agroinfection to improve specific infectivity of the resulting clone is also explained. Curr. Protoc. Microbiol. 7:16F.1.1‐16F.1.26. © 2007 by John Wiley & Sons, Inc.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…The main goal of infectious clone assembly is the construction of plasmid vectors that harbour a faithful copy of virus genomes, which in appropriate conditions can launch a plant infection that mimic natural ones. Detailed step‐by‐step protocols have been described elsewhere (Nagata and Inoue‐Nagata, ; Peremyslov and Dolja, ), and a workflow summary is shown in Figure . Here, we review recent improvements that have allowed one‐step, streamlined assembly of binary infectious clones with no intermediate subcloning steps.…”

“…Proper design of hammerhead ribozymes allows the production of RNA genomes with the desired 3 0 as well as 5 0 ends. Hammerhead ribozymes show robust activity in diverse in vivo systems (Lou et al, 2012), are small and easily incorporated in amplification primers (Jarugula et al, 2018;Pasin et al, 2018;Peremyslov and Dolja, 2007). Viral sequences might interfere with ribozyme folding, leading to reduced or no cleavage.…”

Brief report of the construction of infectious DNA clones of South African genetic variants of grapevine virus A and grapevine virus B

“…Expand reverse transcriptase and Expand long range DNTP pack kits, both from Roche, were used. The cauliflower mosaic virus (CaMV) Ca35S promoter was amplified from binary plasmid pCAMBIA1305.1, and linked to DNA fragment complementary to the 5′ end of the virus genome in PCR described by Peremyslov and Dolja (2007). The oligonucleotide virus-specific and Ca35S-specific primers sequence used in this study were designed manually using the GenBank sequence data, and are shown in Table 1.…”

“…2. Fragments III of GVA P163-M5 and GVB 953-1 were PCR amplified using fragments I and II as templates, and primers pair 5′Ca35S.F1/M5.573.Aat.R and 5′Ca35S.F2/953.880.Age.R, respectively, according to the method described by Peremyslov and Dolja (2007) b Fragment IV of GVB 953-1 was amplified using primers pair 3′Ca35S-5′953.F/953.2704.Avr.R…”

“…2), blinded with T4 DNA polymerase (Thermo Scientific) and cloned to binary vector pCAMBIA1305.1 (11,846 bp) and later to modified binary vector pCB302-NoX-ER-CFP (Peremyslov and Dolja 2007). The pCB302-NoX-ER-CFP vector (6956 bp) kindly donated by Dr. V. Peremyslov (Oregon State University, USA) is a derivative of the mini binary vector pCB302 reported by Xiang et al (1999).…”