Barr, John Nicholas (1993) Expression of the nucleoprotein and phosphoprotein genes of pneumonia virus of mice and specific interactions of the gene products. PhD thesis, University of Warwick.

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Abstract

Following the molecular cloning of the PVM
genome, the opportunity to
the individual
genes and proteins of
PVM has
arisen.
This
study
investigated
nucleocapsid
(N)
gene and the phosphoprotein
(P)
gene of
PVM
and attempted to
characterise the polypeptide products expressed from the N
and
P
genes both in
vitro
in PVM-infected
cells.
The
ability of the PVM N
and
P
proteins to
interact
with
other was also
investigated.
The
nucleotide sequence of the PVM P
gene was
determined to be 903
nucleotides
in length
and shown to comprise a
long
open reading
frame
capable of
encoding the 295
amino acid
long P
protein and also a smaller second
ORF
with the
potential to express a polypeptide
137
amino acids
in length. The PVM P
protein
shows overall amino acid
homology
of
35.3%, 35.6%
and
28.3% to the P
proteins of
pneumovirus members
HRSV, BRSV
and
TRTV
respectively.
The PVM P
gene
contrasts with the P
genes of other pneumovirus genus members which
do
not possess
extensive alternative
ORFs.
Both the N
and
P
genes of
PVM
were shown to be
capable of
directing the
synthesis of more than one polypeptide product
both in
vitro and
in PVM-infected
BSC1
cells. mRNA transcribed from the PVM P
gene
long ORF directed the in
vitro
expression of the 39 kDa P
protein and
four
additional polypeptides.
By
constructing
transcription plasmids that contained
5' terminally truncated P
gene cDNA
insets,
these polypeptides were
determined to be
expressed by translational initiation
on
internal P
gene
initiation
codons.
Western blot
analysis
determined
that in
addition to
PVM P
protein, two of these in
vitro expressed P
protein species, with molecular
weights of
26 kDa
and
23 kDa,
were expressed in PVM-infected BSC 1 cells and this
observation was supported
by the results of anti-P protein monoclonal antibody
epitope mapping studies.
The
ability of the PVM P
gene to direct
the expression of
P
protein related polypeptides
from internal initiation
codons
is
a
feature
not yet
described for
any other pneumovirus member.
By immunising
rats with a synthetic peptide, antiserum specific
for the second
polypeptide product
(P2)
was generated.
Western blot
analysis using this anti-P2
antiserum
identified
a species thought to represent
P2 in PVM-infected BSC 1
cell
material.
The
ability of the PVM P
gene to express a polypeptide
from
an alternative
is
a
feature
common to the P
genes of most other morbilliviruses and
paramyxoviruses.
mRNA transcribed from PVM N
gene cDNA was able to direct the in
vitro
translation of the 43 kDa N
protein and also a
highly
abundant polypeptide with a
molecular weight of
24 kDa
which was shown to be
expressed by
way of
internal
initiation
on the fifth N
gene
AUG
codon of the N
gene sequence. The 24 kDa N
protein related polypeptide was expressed in E.
coli, purified, and used to immunise
a
rabbit
for the production of anti-24
kDa
polypeptide antiserum.
Western blot
analysis
using this antiserum with
PVM-infected BSC1
cells
detected the 43 kDa N
protein, a
highly
abundant
30 kDa N
protein related species, but
not the 24 kDa
polypeptide.
precise
identity
of the 30 kDa
polypeptide was not
determined. Possible
mechanisms which could account
for the expression of the protein products of the N
P
genes are
discussed.
By
using a protein
blotting technique the interaction that occurs
between the N
P
proteins of
PVM
was
investigated. The P
protein
binding
affinities of
in
vitro
expressed truncated N
proteins suggested that many regions of the N
protein are co-
operatively
involved in the binding
process, although some regions contributed more
than others.
The N
protein of
Sendai
virus
is believed to bind to the Sendai
virus
P
protein
in
a similar way.
It
was also
determined that both the amino and the carboxyl-
terminal regions of the PVM P
protein were
found to be
essential
for binding
to N
protein.
This
contrasts with the situation
determined for Sendai
virus
in
which
344 P
protein amino-terminal amino acids were
found to be dispensable for binding N
protein.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Library of Congress Subject Headings (LCSH):	Nucleoproteins , Phosphoproteins, Viral genetics, Viral pneumonia , Pneumonia, Virus diseases -- Research, Genetics -- Research
Official Date:	November 1993
Dates:	Date Event November 1993 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Easton, A. J. (Andrew J.)
Extent:	xiv, 247 leaves
Language:	eng

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