Tiempo de ciclo

The 28 potentially IFN-sensitive viruses selected via FACS analysis were then sequenced in an attempt to identify mutations within the rPIV5mCh genome that could contribute to their inability to block IFN signalling. As it was expected that mutations leading to this phenotype would occur in the viral IFN antagonist V protein, we first set out to sequence the V/P gene of each mutant that encodes both the V and P proteins due to a process known as ‘RNA editing’. Notably, PIV5 V mRNA is a faithful transcript of the V/P gene, whereas the P mRNA contains two non-templated G residues which are added during transcription when the

Isolation of potentially IFN-sensitive viruses using FACS viral polymerase stutters on a specific site, upstream of the insertion site (reviewed in Parks et al 2011). This results in both the V and P proteins containing a common N terminus but each having a unique C terminus.

To sequence the V/P gene of each of the potentially IFN-sensitive virus, Vero cells were infected with each of the viruses at MOI 5 and incubated for 18 hours before RNA extraction. Genomic viral RNA was then reverse transcribed to cDNA using a primer targeted against the sequence flanking the downstream sequence outside of the V/P open reading frame (ORF). This fragment was then PCR amplified, using the same primers and blunt end cloned into the vector pJET 1.2. Colony PCR using primers specific for pJET1.2 was then completed to identify colonies containing the correct insert. Sequencing of the insert was then completed from three individual positive colonies and sequences aligned to the published PIV5 W3 P (AFE48445.1) and V gene sequences (AFE48446.1). Surprisingly, 26 of the 28 viruses sequenced were found to contain the same set of 8 nucleotide mutations, subsequently named rPIV5mCh-α. Notably, each of these mutants displayed the attenuated mutant phenotype 1 during FACS analysis. The other two mutants contained a different set of 2 nucleotide mutations, subsequently named rPIV5mCh-β. Notably, each of these mutants displayed the attenuated mutant phenotype 2 during FACS analysis. Both rPIV5mCh-α and rPIV5mCh-β nucleotide and corresponding amino acid mutations are catalogued in Figure 5.4. To aid visualisation, each of these amino acid mutations was mapped relatively onto the V and P proteins also in Figure 5.4.

Isolation of potentially IFN-sensitive viruses using FACS L51P L102 P L122 P L132 P F135 S Y175 H L51P L102 P L122 P L132 P F135 S V P F144 I F144 I T214 A B) rPIV5mCh-β V P A) rPIV5mCh-α Nucleotide changes in V mRNA Amino acid changes in V protein Nucleotide changes in P mRNA Amino acid changes in P protein

T430A F144I T430A F144I

RNA polymerase GG insertion !

492 and 493 to make P mRNA!

A640G T214A A642G Silent

Nucleotide changes in V mRNA Amino acid changes in V protein Nucleotide changes in P mRNA Amino acid changes in P protein T152C L51P T152C L51P T305C L102P T305C L102P T307C Silent T307C Silent T364C and T365C L122P T364C and T365C L122P T395C L132P T395C L132P T404C F135S T404C F135S

RNA dependent RNA polymerase GG insertion !

492 and 493 to make P mRNA!

T523C Y175H T525C Silent

Common region shared between V/P Unique C terminal region

Isolation of potentially IFN-sensitive viruses using FACS

Figure 5.4: Nucleotide and amino acid mutations in V and P genes/proteins of rPIV5mCh-α and β. The nucleotide and corresponding amino acid mutations in V and P genes/proteins, followed by an illustration of the relative positions of the amino acid mutations in the V and P proteins are demonstrated for rPIV5mCh-α (panel A) and rPIV5mCh-β (panel B).

The mutant rPIV5mCh-α contains eight nucleotide changes from thymine to cytosine in the V/P gene. Specifically, 7 of these nucleotide mutations correspond to 6 amino acid changes within the V protein and 5 amino acid changes within the P protein. Interestingly, the 5 mutations found within the region common to both the V and P gene correspond to 4 amino acid changes from Leucine to Proline (L51P, L102P, L122P and L132P) and the other converts a Phenylalanine to a Serine (F135S). This was somewhat surprising, as we know that amino acid changes to Proline, which is a cyclic amino acid, can dramatically change protein structure. Furthermore we know that the P protein is essential to viral synthesis (Fuentes et al. 2010), yet despite these mutations it appears that this viral mutant can replicate efficiently. The final mutation, which is synonymous in the P protein but non-synonymous in the V protein, mutates Tyrosine to Histidine (Y175H). In contrast, the rPIV5mCh-β mutant contains only 2 nucleotide changes in the V/P gene, one thymine to adenine and one adenine to guanine. These correspond to 2 amino acid changes within the V protein and one amino acid change within the P protein. The mutation found in the common region of the V and P protein mutates Phenylalanine to Isoleucine (F144I). The other mutation, which is synonymous in the P protein but non-synonymous in the V protein,

Isolation of potentially IFN-sensitive viruses using FACS mutates Threonine to Alanine (T214A). Once identified, each of the V protein amino acid mutations were mapped onto the V protein structure, unfortunately no structure has been solved for the PIV5 P protein (Figure 5.5). This highlighted that rPIV5mCh-α and rPIV5mCh-β mutations both appear in structured regions of the V protein such as beta sheets and alpha helices.

In summary, two different mutant viruses were isolated from a stock of rPIV5mCh multiple times and both were found to contain mutations with their V and P proteins. Before we attempted to analyse these viruses further we decided to try to increase the total number of potential mutants isolated whilst adapting this method for use with non-fluorescent viruses. This was important as not all viruses have a readily available recombinant expressing a fluorescent protein and we wanted to create an adaptable method that can be used on multiple viruses.

Isolation of potentially IFN-sensitive viruses using FACS

Figure 5.5: Mapping the positions of amino acid mutations rPIV5mCh-α and rPIV5mCh-β to the wild-type V protein structure (Protein Data Bank accession number 2B5L). The structure of wild-type V protein with the positions of rPIV5mCh-α (panel A) and rPIV5mCh-β (panel B) mutations highlighted (structures adapted using PyMOL (Schrodinger)).

A

Wild-type V protein

highligh2ng posi2on of rPIV5mCh-β muta2ons

Wild-type V protein

highligh2ng posi2on of rPIV5mCh-α muta2ons

Isolation of potentially IFN-sensitive viruses using FACS