Production of Myxoma virus gateway entry and expression libraries and validation of viral protein expression

Abstract

Invitrogen's Gateway technology is a recombination-based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high-throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development.

Figure 14A.2.1

Overall strategy for Gateway recombination cloning of MYXV ORFs. Individual MYXV ORFs are PCR amplified using genomic viral DNA as a template in two PCR steps to generate a product with complete attB sequences flanking gene-specific sequence. This PCR product is incubated with a Gateway donor vector containing the attP sequence in a BP reaction to create an entry clone, which is then sequence verified. Note that the termination codon has been replaced with a leucine codon to allow for C-terminal tag fusions in subsequent Gateway expression vectors. The entry clone is incubated with pANT7-cGST expression vector in a LR reaction to generate an expression clone with the C-terminal GST tag; GST-tagged protein expression is verified using the TNT Coupled Reticulocyte Lysate System (Promega) and immunoblot analysis. Adapted from Park and LaBaer (2006) and Invitrogen (http://www.invitrogen.com/gateway).

Figure 14A.2.2

Two-step PCR strategy. A first round of PCR is performed using MYXV genomic DNA as template and primers with gene specific and partial att sequence. A second round of PCR is then performed using the first-round PCR product as template, amplifying with universal att primers that generate a product containing the complete attB recombination sequence. The att sequence added to the gene-specific portion of the PCR product by successive rounds of PCR with these primers is indicated by the shaded areas. Adapted from Park and LaBaer (2006).

Figure 14A.2.3

Primer sequence. For gene-specific primers, the partial att sequence is shown in capital letters. For the forward primer, Kozak sequence (CACC) precedes the ATG, which is followed by 15 to 30 nucleotides of viral gene sequence. In the reverse primer, the stop codon is replaced by the leucine anticodon (CAA) since all ORFs are ultimately cloned into a C-terminal GST expression vector (pANT7-cGST) to generate C-terminal fusion proteins. Adapted from Park and LaBaer (2006).

Figure 14A.2.4

PCR amplification products prior to gel purification. (A) First-round PCR products for representative samples. (B) Second-round PCR products amplified from first-round PCR products shown in panel A. Although a full-length 6-kb product was obtained for M134A in a first round of PCR, this ORF did not amplify with universal primers for a second round of PCR. ORFs that did not amplify are indicated with an asterisk (*).

Figure 14A.2.5

Protein expression for representative MYXV GST-tagged ORFs M001L-M066R. Those which did not express are indicated with an asterisk (*). Size markers are shown in kDa. Note that although GST-M047R and GST-M065R did not appear to express, protein expression was verified in a later immunoblot analysis. Furthermore, some expressed GST-fusion proteins migrate differently than what was predicted from the viral ORF.

Figure 14A.2.6

Protein expression for representative MYXV GST-tagged ORFs M067L-M147R. Those which did not express are indicated with an asterisk *. Size markers are shown in kDa. Note that some expressed GST-fusion proteins migrate differently than what was predicted from the viral ORF.