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heehawultraMechanics

Feb 22, 2014 (3 years and 7 months ago)

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Flaviviridae

Positive stranded RNA viruses

Flaviviridae


Enveloped
virions

made up of a lipid
bilayer

with two or more types of envelope (E)
glycoproteins

surrounding a
nucleocapsid
.


Single
-
stranded, positive
-
sense genome RNA
complexed

with multiple copies of a small,
basic
capsid

(C) protein.


Life Cycle


Binding
and uptake
involve receptor
-
mediated
endocytosis

via cellular
receptors
specific for
viral envelope proteins.


Low
pH of the
endosomal

pathway
induces fusion of the
virion

envelope
with cellular
membranes.


Following
uncoating

of the
nucleocapsid
, the
RNA genome is release into
the cytoplasm.


The viral
genome serves three discrete roles within the life cycle:


mRNA
for translation of all
viral proteins
,


template
during RNA replication, and


g
enetic material
packaged within new virus particles.


Viral proteins
are produced as part of a single long
polyprotein

of
more
than 3,000 amino acids that is cleaved by a
combination of
host and viral
proteases.


The
structural
proteins
are encoded in the N
-
terminal portion of the
polyprotein

with
the
nonstructural

(NS) proteins in the
remainder.


RNAreplication

occurs in
cytoplasmic

replication
complexes via
synthesis of a genome
-
length
minus
strand RNA intermediate
.


Progeny
virions

are thought to
assemble by
budding into an intracellular membrane
compartment
, most
likely the endoplasmic
reticulum (ER), then
transited through
the host
secretory

pathway and released at the
cell
surface.

Examples


Dengue virus


Hepatitis C Virus

Attachment


Flaviviruses

can utilize multiple receptors
for
different cell types and in
different host species.


Infection of
dendritic

cells (DC) is particularly important
because these
intradermal

cells can be primary targets
early in
infection
.


Other proteins
identified
as
receptors include:


αvβ3
integrin
,


GRP78
(
BiP
),


and
CD14


In
addition, highly
sulfated

glycosaminoglycans

(e.g.,
heparan

sulfate
) have
been shown to play an important
role in
the initial attachment of several
flaviviruses

to
target cells.


Virus
particles
opsonized

with
immunoglobulins

show
enhanced binding
and infection
of cells
expressing immunoglobulin
Fc

receptors. It is
widely
suspected that antibody
-
enhanced infection
may be
relevant to the
pathogenesis of DF and DHF, which
occur more
frequently in people
previously exposed to
other DENV serotypes.

Entry


Internalized
via
clathrin
-
coated
pits and
trafficked to a
prelysosomal

endocytic

compartment where
low pH induces fusion
between the virus
and host
cell membranes to
release the virus
nucleocapsid


It
appears that viral
genomes are
directly
accessible for translation after membrane
fusion.

Dengue Virus


Genomes consist
of a single, positive
-
strand RNA of
≈11 kb (sedimentation
, 42S
)


5 ‘cap
, m7GpppAmpN2


Additional
methylation

of the N2 residue (
type II
cap)
has also been detected in RNA from
infected cells
.


Unlike
cellular messenger RNA (mRNA),
flavivirus

genomes lack a 3
polyadenylate

tail.


Genomes encode a
single long open reading frame
(ORF) flanked by
5 and
3
noncoding

regions (NCR) of
≈100 nucleotide (
nt
) and
400 to 700
nt
,
respectively.

RNA Replication


Replication begins
with the synthesis of a
genome
-
length minus
strand RNA
, which then
serves as a template for the synthesis
of
additional
plus strand RNA.


Minus
strand RNA has
been detected
as early
as 3 hours after infection

Hepatitis C Virus (HCV)


The HCV genome is an uncapped,


9.6
-
kb
RNA
containing highly
structured 5 and 3
ends.


The
5 NCR is
a well
-
conserved
, 341
-
nt sequence
element that folds
into a
complex structure
consisting of four major domains
and a
pseudoknot
.


The
first 120
nt

serves as
a minimal
replication
element, although nearly the entire
5 NCR
is
needed for efficient RNA
replication.

RNA Replication



Translation
and RNA replication are mutually
exclusive
processes
, because they proceed in opposite
directions on
a given RNA template.


Positive
-
strand
RNA
viruses regulate
the rate of genome
translation
versus replication
.


Translation
of the HCV genome
occurs more
frequently
than replication.


One
way this could
be regulated
is via crosstalk between
the determinants
that control
translation and genome
replication.


E.g

the
cellular PTB protein binds to the HCV 5 NCR
and
core
coding region where it may modulate IRES
activity
and
to the 3 end where it may repress
replication.


It has been suggested that HCV
translation can
be
autoregulated

through product
inhibition.


Low
levels of HCV core protein can enhance HCV
IRES mediated translation
, whereas high
concentrations
inhibits HCV translation


Polycytidine
-
binding
protein 2 (PCBP
-
2) binds to
the HCV
5
NCR also noted in polioviruses that
PCBP binds 5’ NCR and
RdRP

of
poliovirus, to
control the switch between translation and
replication.


Location of Replication


The
HCV
genome is
recruited out of
translation and into a
membrane associated
replication
complex, or
replicase
.


Specifically, NS
proteins and HCV RNA
associate with a
dense
perinuclear

matrix
of≈85
-
nm vesicles
termed the membranous
web.



HCV RNA replication is stimulated by
increased availability
of saturated and monounsaturated fatty acids
, and
inhibited by
polyunsaturated fatty acids or inhibitors
of
fatty
acid
synthesis.


HCV RNA is
protected from
nuclease degradation by a
detergent
-
sensitive
membrane suggesting
that RNA
synthesis
may be
enclosed within the membranous web.


Similarly
,
replicase

activity
is insensitive to protease
digestion unless
solubilized

by
detergent, whereas most of
the NS proteins
are digested
by this treatment
.


These
data
support the
hypothesis that active
replicase

is
bound by a
limiting membrane
and demonstrate that a vast
excess of
NS proteins
are produced.