FIGURE 8. The effects of overexpression of C14orf166 proteins
on influenza A WSN and DkPen virus RdRp activity. The HEK293 cells were
grown in a 24-well plate and co-transfected with a 200 ng of
pCHA-C14orf166 expression plasmid and influenza A virus mini-replicon
plasmids, and the reporter enzyme activities were defined as previously
described23.
DISCUSSION
The C14orf166 protein is referred to as RNA
Transcription,Translation and Transport Factor (RTRAF) according to
recent data. This protein is a nucleo-cytoplasmic protein having a
binding activity to RNA molecules and the RNA polymerase II complex31. As a result of these activities, it has a positive
regulatory effect on RNA Pol II transcription and is involved in
processes related to RNA metabolism such as tRNA splicing as part of the
tRNA splicing ligase complex. In addition, it is thought that it also
functions in RNA transport. The C14orf166, a multifunctional protein
with these properties, is associated with some cancer types. It has been
suggested that the overexpression of C14orf166 has a role in the
development of non-small-cell lung cancer (NSCLC), and it would be
beneficial to consider it in the treatment of high-risk
patients32. It has been reported that this protein is
overexpressed in breast cancer33. The expression of
both the C14orf166 mRNA and protein was also found to be upregulated in
cervical cancer cell lines and tissues18. Therefore,
it has been suggested that it can be used as a biomarker for the
diagnosis and prognosis of some cancer types34.
It has been determined that the C14orf166 protein, which has important
functions in cellular RNA metabolism, also participates in the
replication/transcription processes of some viruses. The C14orf166
protein interacts with the hepatitis C virus core protein and
upregulates the viral infection22. Another important
virus type with which the C14orf166 protein is associated is the
influenza A virus. It has been stated that this protein binds to the
influenza A virus RbRP enzyme PA subunit and stimulates the virus
replication21. Our results with the minireplicon
models also showed that C14orf166 stimulates the influenza viral
polymerases (Figure 8).
In this study, we demonstrated that the full-size and truncated
C14orf166 proteins in different sizes interacted with the influenza
virus PA protein in the Y2H assay using the PA protein as a bait and the
HEK293 Matchmaker cDNA (Figures 1 and 2). Among these proteins, the
smallest fragment that interacts with PA protein consists of 69 amino
acid residues that form the carboxy-terminal end of the C14odf166
(AD-C69). Therefore, it was concluded that the carboxy-terminal end of
C14orf166 is important and sufficient for interaction with the viral PA
protein. In a previous report, it was shown that the C14orf166 protein
interacts not only with PA alone but also with the influenza virus RdRP
complex and co-localizes with viral
ribonucleoproteins12. Thus, the C14orf166 protein is
not thought to bind to the regions of PA that interact with PB2 and PB1
subunits. In the Y2H assay where we used the amino-terminal half of the
viral PA protein as a bait, no C14orf166 protein could be detected among
the host interactor proteins, which led us to conclude that the
amino-terminal region of PA is most likely not a target for this
protein. Therefore, we focused on the carboxy-terminal moiety of PA for
binding of the C14orf166 protein in docking analyses. Although the most
probable 3D structures were selected in terms of free energies among the
AD-C14orf166 protein models interacting with PA, these models may not
reflect the real structures of the proteins. Here, we focused on the
C14orf166:PA interaction model rather than the 3D structures. The
docking results of the PA with the predicted 3D structures of the C69
peptide (non-fused with GAL4-AD) and AD-C69 (fused with GAL4-AD)
expressed in the yeast cells indicated that this peptide/protein binds
to the ligand (peptide) recognition site6 located in
the carboxy-terminal region of the
PA. In docking models, it was
observed that C69 and AD-C69 clustered in this region, mostly covering
the 610th to 630th amino acid residues on the PA (Figures 5A, 6A, and
6C). The docking results of AD-C209:PA and AD-C244+4:PA proteins
synthesized in yeast cells showed that the carboxy-terminal ends of
these fusion proteins have a high affinity for the peptide binding site
of the PA protein such as C69 and AD-C69 (Figure 7). One the docking
models of AD-C244+4, which involves full-length C14orf166 and the PA
protein, the prominent amino acids in the interaction areas of these two
were evaluated with PyMol software and PDBsum algorithm. In this model,
there is the possibility of forming three salt bridges and fourteen
hydrogen bonds between AD-C244+4 and the PA protein (Figure 7 E).
Although the amino acid residues in the interaction area of the PA
protein show a distribution within the 450th to 700th amino acids, the
intense interaction site is seen in the 610th to 630th amino acid
positions. Huarte et al., as a result of their mapping study, suggested
that amino acid residues in positions 493 to 512 and 557 to 574 on the
PA protein are important for binding to C14orf16621.
Although this region of PA agrees with our results, docking analysis
data point to amino acids within the 610th to 630th position as the most
intense interaction site in our models.
In conclusion, possible interaction models between influenza A virus PA
and human C14orf166 protein were revealed by reconciling the
experimental results of the Y2H assay carried out with the influenza A
virus PA as a bait, existing literature information, and the results of
in silico 3D prediction/protein-protein docking analysis. It is very
difficult to accurately predict protein-protein interaction patterns
similar to those in the natural environment with in silico tools
ignoring the factors in the cellular environment. It was concluded that
the data of the Y2H assay will make an important contribution to
predicting the 3D structures and possible interaction models in a form
close to its native structure. The interaction models presented here for
human C14orf166 and viral PA protein will allow us to better understand
the influenza A virus replication and virus-host relationship.