Polymerase PB1-PB2 subunits from Influenza A Virus
Brittany Currey '11 and Lauren Brady '11
influenza virus belongs to Orthomyxoviridae
family, which is composed of six different RNA viruses.
influenza A virus contains eight
negative-strand RNA (vRNA) segments,
which encode 10 different viral proteins. This is a particularly common
in birds, mammals, and humans.
estimated death toll in the United States on average is 50,000 people
Given the more recent
outbreaks with avian influenza, particularly in Asia, many fear that
strands will adapt to human hosts, as this has
been the cause of
influenza pandemics in the last 300 years. Symptoms
headache, and nasal discharge which can give way
to more obstructive pulmonary and heart problems including cardiac
In the search for new
pharmaceuticals, the viral RNA (vRNA) polymerase has the potential to
strong target of study due to high conservation among strains of
vRNA polymerase is composed of three
PA. Although there is contact between PA and PB1, there are no direct
interations between PA and PB2. These
subunits play distinct roles within the polymerase and are all crucial
viral transcription and replication. The
PB2 subunit of the RNA polymerase forms a ribonuleoprotein (RNP)
its eight genome segments and moves into the nucleus. Once in the
RNP complex initiates the process of cap snatching before viral mRNA
moves to the cytoplasm to undergo translation. During
this process, PB2 binds cap-containing
mRNA in order to produce primers for RNA synthesis.
endonuclease from the PA subunit then cuts the
cap-containing oligonucleotide pre-mRNA of the host cell
allowing it to
extended into viral mRNA by the polymerase. (Cap Snatching)
the RNAP complex, the PB1-PB2
interface is needed for transcription initiation and is dependent on
N-terminal fragment of PB2
studies suggest that suitable small molecules may
disrupt this interaction and possibly restrict viral replication,
making this easily accessible site a strong drug target.
co-precipitation assay was used to observe the interaction between the
C-terminus of PB1
(PB1-C) and the N-terminus
1-37 and 1-86 of PB2-N are required for subunit binding to
PB1-C. A stable RNAP
was crystallized consisting of residues
678-757 of PB1-C and
residues 1-37 of PB2-N.
are two copies of PB1
The majority of interaction energy is provided by helix 1 of
against helices 2
and 3 of
of PB1-C is positioned
between all three
helices of PB2-N.
interactions, hydrogen bonds, and some
buried apolar contacts between PB1-PB2 allow for the complex to remain
the RNAP, the
interface contains the most extensive buried surface area
as a result
of the '3
plus 3' helix structure. The interface between PB1-C and PB2-N contains
bridges between Glu
2 and Lys 698
3 and Asp 725
3 and Lys 698
and Lys 698
include an interaction between two PB2 residues (Ile
4 and Leu 7).
between polypeptides supply further stability of
and PB2 double mutants
presence of PB2 is necessary for
functionality of RNAP.
shape of PB2 hinders most intermolecular contacts between its three
deletions of Helix
its importance as measured by vRNA synthesis, reducing production of
product by 90%.
When two nonpolar amino
and Leu 7,
or Leu 7
with polar serine residues, polymerase activity was greatly reduced. Similar
replacements in PB1 (Val 715
746, or Ile 746
serine residues not only led to the
in vRNA but also cRNA and mRNA. The
polar residues at the protein surface (Ser 713
a serine deep within the hydrophobic core without preventing PB1 and
amino acid replacements on PB1
significantly prevent PB1-PB2 binding enough to inhibit mRNA production. The
only exception was the replacement of Val 715
a Ser (V715S) which still showed significant binding to PB2
reduction in RNAP activity. This
mode of interaction has an effect on the efficiency of polymerase. Another
single mutant displaced Leu 695
an aspartate without preventing PB1-PB2 binding due to
accessibility to solvent
allow for the
functionality of viral RNA polymerase. Helix
that dramatically affects the activity of
the RNAP when removed.
V715S mutation does not inhibit the
interaction between PB1 and PB2 but hinders the communication between
PB2 resulting in the loss of activity of RNAP. The
PB1-PB2 may be a
potential target for novel anti-influenza drugs due
to its importance in viral replication.
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