RNA
Polymerase PB1-PB2 subunits from Influenza A Virus
Brittany Currey '11 and Lauren Brady '11
Contents:
I.
Introduction
The
influenza virus belongs to Orthomyxoviridae
family, which is composed of six different RNA viruses.
The
influenza A virus contains eight
negative-strand RNA (vRNA) segments,
all of
which encode 10 different viral proteins. This is a particularly common
disease
in birds, mammals, and humans.
The
estimated death toll in the United States on average is 50,000 people
annually.
Given the more recent
outbreaks with avian influenza, particularly in Asia, many fear that
these
viral
strands will adapt to human hosts, as this has
been the cause of
three
influenza pandemics in the last 300 years. Symptoms
include fever,
headache, and nasal discharge which can give way
to more obstructive pulmonary and heart problems including cardiac
failure and
bacterial pneumonia.
In the search for new
anti-influenza
pharmaceuticals, the viral RNA (vRNA) polymerase has the potential to
be a
strong target of study due to high conservation among strains of
influenza
virus.
This
vRNA polymerase is composed of three
subunits: PB1,
PB2,
and
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
for
viral transcription and replication. The
PB2 subunit of the RNA polymerase forms a ribonuleoprotein (RNP)
complex with
its eight genome segments and moves into the nucleus. Once in the
nucleus, 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.
An
endonuclease from the PA subunit then cuts the
cap-containing oligonucleotide pre-mRNA of the host cell
allowing it to
be
extended into viral mRNA by the polymerase. (Cap Snatching)
Within
the RNAP complex, the PB1-PB2
interface is needed for transcription initiation and is dependent on
the short
N-terminal fragment of PB2
(PB2-N) .
Recent
studies suggest that suitable small molecules may
disrupt this interaction and possibly restrict viral replication,
making this easily accessible site a strong drug target.
II. PB1-PB2
interaction Domain
A
co-precipitation assay was used to observe the interaction between the
C-terminus of PB1
(PB1-C) and the N-terminus
of PB2
(PB2-N) .
Residues
1-37 and 1-86 of PB2-N are required for subunit binding to
PB1-C. A stable RNAP
complex
was crystallized consisting of residues
678-757 of PB1-C and
residues 1-37 of PB2-N.
There
are two copies of PB1
and
PB2
subunits,
each containing
three
α helices.
The majority of interaction energy is provided by helix 1 of
PB2-N,
which lies
against helices 2
and 3 of
PB1-C.
Helix
1
of PB1-C is positioned
between all three
helices of PB2-N.
Polar
interactions, hydrogen bonds, and some
buried apolar contacts between PB1-PB2 allow for the complex to remain
tightly
bound.
Within
the RNAP, the
PB1-PB2
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
four salt
bridges between Glu
2 and Lys 698
,
Arg
3 and Asp 725
,
Arg
3 and Lys 698
,
and Glu
6
and Lys 698
.
Apolar
contacts
include an interaction between two PB2 residues (Ile
4 and Leu 7).
Eight
hydrogen bonds
between polypeptides supply further stability of
the complex.
III. PB1
and PB2 double mutants
The
presence of PB2 is necessary for
functionality of RNAP.
The extended
shape of PB2 hinders most intermolecular contacts between its three
helices.
However,
deletions of Helix
1 reveal
its importance as measured by vRNA synthesis, reducing production of
RNA
product by 90%.
When two nonpolar amino
acids (Ile
4
and Leu 7,
or Leu 7
and Leu
10)
of
helix
1 were
simultaneously replaced
with polar serine residues, polymerase activity was greatly reduced. Similar
replacements in PB1 (Val 715
and Ile
746, or Ile 746
and Ile
750)
with
serine residues not only led to the
reduction
in vRNA but also cRNA and mRNA. The
nearby
polar residues at the protein surface (Ser 713
and Arg
754)
can
easily
accommodate
a serine deep within the hydrophobic core without preventing PB1 and
PB2 binding.
IV. PB1
single mutants
Single
amino acid replacements on PB1
did not
significantly prevent PB1-PB2 binding enough to inhibit mRNA production. The
only exception was the replacement of Val 715
with
a Ser (V715S) which still showed significant binding to PB2
with corresponding
reduction in RNAP activity. This
altered
mode of interaction has an effect on the efficiency of polymerase. Another
single mutant displaced Leu 695
and Ile
750
with
an aspartate without preventing PB1-PB2 binding due to
their
accessibility to solvent
water.
V.
Conclusion
PB1
- PB2
interactions
allow for the
functionality of viral RNA polymerase. Helix
1
of PB2
is
an important
subunit
that dramatically affects the activity of
the RNAP when removed.
The
V715S mutation does not inhibit the
interaction between PB1 and PB2 but hinders the communication between
PB1 and
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.
VI.
References
Boivin,
Stéphane, Stephen Cusack, Rob Ruigrok, and Darren J. Hart.
"Influenza A Virus Polymerase: Structural Insights into Replication and
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Sehr, Peter; Lewis, Joe;
Ruigrok, R.W.H.; Ortin, Juan; Hart, Darren J.; Cusack, Stephen. 2008.
The
structural basis for cap binding by influenza virus polymerase subunit
PB2.
Nature Structural & Molecular Biology 15 (5): 500-506.
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<http://pathmicro.med.sc.edu/mhunt/flu.htm>.
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