Biology Reference
In-Depth Information
short time when compared to the linear peptides [27, 28]. Small peptide molecules
have been used in the development of peptide based vaccines for melanoma [29], in-
hibitors against HIV [30], Dengue and West nile virus [31] and anti-angiogenic in the
treatment of angiogenesis related diseases [32].
As whole virus particles were used in biopanning experiments, in principle, the
selected peptides might interact with any of the three surface proteins such as HA, NA
and M2. Since these inhibitory peptides possess strong anti-viral activity when used
at pre-infection not at post-infection and also inhibit the hemagglutination, it can be
deduced that the peptides (NDFRSKT and CNDFRSKTC) prevent the viral replica-
tion by inhibiting the attachment or entry of the virus into the target cells. There are
many studies on the targeting of the conserved region of the HA protein. Recently,
Jones et al. [33] identifi ed that a well known cell-penetrating peptide, derived from
the fi broblast growth factor 4 (FGF-4) signal sequence, possesses the broad-spectrum
anti-infl uenza activity, which act by blocking the entry of virus through the HA protein
interaction.
Neuraminidase (NA) is the second most abundant surface protein and responsible
for the neuraminidase activity of the virus. It is important both for its biological activ-
ity in removing sialic acid from glycoproteins and as a major antigenic determinant
that undergoes variation. At present, the NAIs such as zanamivir and oseltamivir are
preferentially used for the treatment and prophylaxis of infl uenza [9], as the NA pro-
tein is less mutative when compared with HA. There are three receptor binding sites,
two at the distal ends of both HA subunits and the third one in the NA protein [34]
and changes in both HA and NA glycoproteins will affect the fi tness of the virus [35];
therefore, the effect of peptide on the neuraminidase protein was assessed. Unfortu-
nately, this experiment showed a negative result for the fusion phages and cyclic pep-
tides and partial inhibition result at very high concentration of linear peptide (~35%
inhibition at 1,000 μM). The latter inhibition may be nonspecifi c due to the increased
ability of the linear molecules to attain a structure that facilitates the binding with NA
molecule or merely based on hydrophobicity and charge.
The HA-P1 and NA-P1 interaction was further analyzed by the yeast two-hybrid
system and co-immunoprecipitation. There has been a problem in amplifying the full
length clone of
HA
gene for the past few years in our laboratory. The same problem
has also been reported in few other laboratories working with the same strain in this
region. The 3' end of the vRNA could not be amplifi ed either by primer designed for
conserved region or gene specifi c region based on other similar strain's sequence. The
HA protein should be cleaved into two disulfi de linked HA
1
and HA
2
in order to be
infectious. The C-terminal HA
2
region is very important as it accounts for the entry of
the virus into the host cell and thus serves as a fusion protein [36]. Therefore, the trun-
cated HA protein representing C-terminal end (278 amino acids) of the full length HA
protein was used for the yeast two-hybrid and co-immunoprecipitation experiments.
The yeast hybrid assay turned positive for the both HA and NA proteins although the
β-galactosidase activity for HA is nearly seven fold higher than the NA. Although,
there was negligible or no interaction between NA and P1 as per the results of NA
inhibition test and co-immunoprecipitation results, the yeast two-hybrid experiment
