ISOTIPO/LOGOTIPO (PROTOTIPOS)
ESTUDIO DE REQUERIMIENTOS DE EQUIPO Y HERRAMIENTAS DE TRABAJO
Two PB1-F2 deletion viruses were engineered (overview in Table 3.4).
Virus Mutation Protein levels Phenotype
∆AUG T120C PB1: WT small plaques C153C PB1-F2: null attenuationin vitro
G291A N40: overexpression attenuationin vivo
F2-11 C153G PB1: WT
G291A PB1-F2: null WT phenotype N40: WT
Table 3.4: Summary Chapter 3: Introduced mutations into PB1 abolished PB1-F2
ORF. Whereas the PB1 level appears unaffected for both strains, ∆AUG viruses
overexpress N40. This led to a major attenuation bothin vitroandin vivo.
The removal of the start codon of PB1-F2 had a dramatic effect on the virus both in vitro and in vivo. The only difference between ∆AUG viruses and F2-11 is the altered level of N40, which seems to have detrimental effects on plaque size, growth and especially on virulence. This findings are important as many previous studies to determine the function of PB1-F2 are based on the deletion of the PB1-F2 ORF by removing the start codon. Here it was shown that N40 needs to be taken into consideration when designing PB1-F2 mutants.
CELLULAR POLYMERASE II
DEGRADATION
Aims of Chapter 4
The viral RNA dependent RNA polymerase was reported to bind to the largest subunit of cellular RNA polymerase II (Engelhardt et al., 2005). This interaction resulted in degradation of the hypophosphorylated form of the cellular polymerase. Two single point mutations within the viral polymerase subunits were tested for their influence on degradation and host cell protein shut-off.
The first mutation was located in the polymerase subunit PB2 (V504I), a second mutation was introduced in the polymerase subunit PA (L550I). The effects of the individual single point mutations were analysed as well as the effect of a double mutation.
4.1
Interaction of the influenza A virus RdRp
and cellular RNA polymerase II and the
consequences of this interaction
Cellular mRNA synthesis is performed by a multiprotein complex called RNA polymerase II (RNAP II). The carboxy-terminal domain (CTD) of the largest subunit plays a major role during the transcription cycle. It contains over 50 repeats of a heptapeptide YSPTSPS, which are differentially phosphorylated during the transcription process (Figure 4.1). Upon phosphorylation of serine-5 residues, the RNAPII initiates transcription. Following further phosphorylation at serine-2 residues, transcription proceeds into elongation (Komarnitsky et al., 2000). Coupled with these different phosphorylation stages are varying affinities for transcription factors and factors involved in 5’ and 3’ end processing of pre-mRNAs (Schroeder et al., 2000; Komarnitsky et al., 2000; Kim et al., 1997; McCracken et al., 1997).
Figure 4.1: Differential phosphorylation of the CTD of cellular RNAPII: The hypophosphorylated form of the CTD gets phosphorylated at serine-5 residues which initiates transcription. Proceeding into elongation is realised by serine-2 phosphorylation. Taken from Phatnani and Greenleaf (2006).
Although influenza A viruses encode their own RNA-dependent RNA polymerase (RdRp), it was assumed for a long time that viral replication depends on a functional cellular RNA polymerase II. Two events in the viral life cycle are thought to be coupled to cellular transcription:
cellular mRNAs (Bouloy et al., 1978; Plotch et al., 1981).
(2) Influenza A virus segments 7 and 8 produce spliced mRNAs, and splicing is performed by the cellular splicing machinery (Inglis et al., 1979; Lamb et al., 1981).
Viral RdRp was found to interact with the largest subunit of cellular RNAPII (Engelhardt et al., 2005). More specifically, this interaction was shown to be between the viral polymerase trimer and the C-terminal domain (CTD). None of the polymerase subunits alone nor one of the dimers were able to bind to and degrade the RNAPII. Interaction was confirmed between the RdRp and the CTD in its hyperphosphoryated form, more specifically with the Serine-5-phosphorylated form (Engelhardt et al., 2005). It is believed that this provides the viral polymerase with 5’ capped pre-mRNAs that are needed to initiate viral transcription. However, following interaction, the largest subunit of the RNAPII in its hypophosphorylated form was shown to be degraded (Rodriguez et al., 2007).
When this project was started, the involvement of the individual viral polymerase segments was not known. Hence, the impact of PB1-F2 was determined by evaluating the degradation of RNAPII in rWSN WT or rWSN
∆AUG infected cells. Specific antibodies were used to differentiate between the different phosphorylated forms of the CTD (Table 4.1).
Antibody Epitope Recognition
8WG16 Non-phosphorylated Non-phosphorylated repeats H5 Ser2-P Ser-2P, Ser5-P, Ser2-P + Ser5-P
Table 4.1: Anti-CTD monoclonal antibodies recognising specific epitopes depending on their phosphorylation state. (Jones et al., 2004; Rodriguez et al., 2007)
Confluent monolayers of MDCK cells were infected with one of the two viruses and cells were lysed at the indicated time points. Results of the western blot analysis are shown in Figure 4.2. Consistent with previous reports (Rodriguez et al., 2007), a decrease in the accumulation of the hypophosphorylated RNAPII in rWSN WT infected cells was observed from 6 hpi onwards. In contrast, no decrease was observed in the accumulation level of Serine-2-phosphorylated RNAPII, detected by the antibody H5. The degradation correlated with the onset of viral protein synthesis. No difference was observed in cells infected with the rWSN ∆AUG virus,
0 0 2 4 6 8 10 12 12 0 0 2 4 6 8 10 12 12
MOCK
MOCK rWSN WT MOCK rWSN ΔAUG MOCK
hpi 8WG16 H5 PB1-F2 PB1 α-tubulin
Figure 4.2: Degradation of cellular polymerase II did not depend on PB1-F2:
Confluent monolayers of MDCK cells were infected with rWSN WT, rWSN ∆AUG
or Mock infected at an MOI of 3 and cells were lysed at the indicated time points. 8% Bis-Tris gels were used to detect the cellular polymerase using the antibodies 8WG16 and H5. 4 - 12% gradient Bis-Tris gels were used for PB1 detection, whereas 16% Tricine-SDS-PAGE was used to detect the small hydrophobic protein
PB1-F2. α-tubulin was used as an internal loading control.
More recently the involvement of PB2 and PA were suggested, when comparing low-virulence (lvPR8) and high-virulence (hvPR8) strains of A/PR/8/34 for their ability to degrade the largest subunit of RNAPII (Rodriguez et al., 2009). It was shown that the degradation was restricted to non-attenuated viral strains, because the lvPR8 and a cold-adapted strain (A/Ann Arbor/6/60) were found not to degrade the cellular polymerase. When comparing recombinant viruses, segment 1 and 3 gene products were identified to contribute to the degradation whereas segment 2 did not have an influence.
It is not fully understood how the interaction between cellular RNAPII and viral RdRp is maintained. A recent study suggested the kinase cyclinT1/CDK9 to function as a mediator between the CTD and the viral polymerase complex (Zhang et al., 2010a). CDK9 was shown to bind to all three polymerase subunits, PB2, PB1 and PA, and the interaction promoted viral transcription. Cyclin T1/CDK9 phosphorylates serine-2 residues in the CTD of the large subunit (Marshall et al., 1996). Previously, binding was especially suggested between RdRp and the Ser-5P form of the CTD (Engelhardt et al., 2005), but phosphorylation of serine-5 residues is performed by a different kinase (TFIIH). If the viral polymerase complex binds directly to the CTD with Serine-5-phosphorylation or if this interaction