RNA Viruses
I. General
Characteristics
-Three subtypes
a. (+)
b. (-) Strand
c. Double
-The
genome of all RNA viruses except retroviruses encode an RNA-
dependent
RNA polymerase.
-Transcription
of viral genomic RNA and mRNA are not inhibited by
Actinomycin D, which inhibits cellular DNA-directed
RNA synthesis.
-RNA-directed RNA synthesis:
a.
RNA synthesis initiates and terminates at specific sites in the template
b. Catalyzed by virus-encoded polymerases
c. May require viral and/or host accessory
proteins
d.
Most RNA-dependent RNA polymerases initiate synthesis denovo but some require a
primer with a free 3’OH end complementary to the template strand
e. RNA primers may be protein linked and may
contain a 5’ cap
f. Elongation is in a 5’ to 3’ direction
-RNA-dependent RNA polymerases
a.
The active site of most (+) strand, double strand and segmented
(-) strand viruses have the motif Gly-Asp-Asp
b.
The active site of most (-) strand non-segmented viruses have
the motif Gly-Asp-Asn
c.
Structure is analogous to a right hand consisting of a palm (active site),
fingers and thumb.
-RNA molecules contain secondary structures important for
Synthesis,
Translation, and Assembly
a. Stem loop regions
b. Hairpin loops
c. Bulge loops
d. Interior loops
e. Multibranched loops
f. Psuedoknot
-Accessory Proteins in RNA-dependent RNA synthesis
a. Viral proteins
-Viral proteins may have various
roles:
i. Direct the
polymerase to the correct intracellular site
ii. Targets to the
correct initiation site on RNA
templates
iii. RNA helicases
-Unwind
genomes of double stranded RNA
viruses
-Unwind
secondary structures in template
RNAs
-Prevent
base-pairing between template and
nacent strands.
b. Cellular proteins
-Viruses may require different
cellular proteins
-Initiation mechanisms of RNA synthesis
a.
Most RNA-dependent polymerases do not require a primer
except:
i. Picornoviuses
ii. Influenza viruses
iii. Bunyaviruses
b. Two types of Priming Mechanisms
i. Protein Priming
-Picornoviruses
-Primer is a genome
linked protein
ii. Priming by capped RNA
fragments
-Influenza viruses
and Bunyaviruses
-Capped RNA primers
acquired from cleavage of host
cell RNA polymerase II transcripts: Cap
Snatching
-A cap-dependent
endonuclease that is part of the
RNA polymerase creates 10 to 13
nucleotide
long capped primers that serve as primers for
initiation of viral mRNA synthesis.
-Inhibited by the
fungal toxin a-amantinin
-Conformational Changes and Regulation of RNA synthesis
a. Alterations in viral RNA polymerases may
influence template
recognition and initiation:
i. Conformational changes
ii. Proteolytic changes
-Synthesis of (+) strand and (-) strand is not balanced
a. The efficiency of initiation of RNA
synthesis at the 3’ end of the
(+) and (-) strands may be different
b. The polymerase that synthesis one type of
strand may be limited
in its
expression during infection
-Most viral mRNAs contain a terminal poly-A tail
-Mechanisms for switching from mRNA synthesis to genome
RNA
synthesis
a. Picornovirus mRNA is identical to genome
RNA.
b. Different RNA polymerases for mRNA
synthesis and genome
replication
c. Suppression of intergenic stop-start
reactions by nucleocapsid
proteins
d. Suppression of termination by Stem-loop
structures
e. Different templates used for mRNA
synthesis and Genome
replication
f. Suppression of polyadenylation
-Origins of Diversity
a. Misincorporation of nucleotides
-RNA-dependent RNA polymerases
can’t proofread
b. Segment Reassortment
c. RNA recombination
i. Base-pair dependent
ii. Base-pair independent
iii. Base-pair assisted
II. Positive strand RNA viruses
-Virions lack a viral RNA polymerase
-The deproteinized
RNA molecules of these viruses are infectious
and can
be translated in cells to produce viral proteins
-Most lack an envelope
-Genomes are not coated with viral proteins in the virion
-RNA synthesis takes place on internal membranes in the
cytoplasm
i. May allow concentration of replication
components
ii. May allow sufficient packaging of progeny
RNA into virions
iii. May provide lipid components or support
III. Negative
-Virions must contain and RNA polymerase
-Incoming RNA is
not infectious and cannot be copied or translated by
cellular
proteins
-Most are enveloped
-The genomes are organized into nucleocapsids
a.RNA-dependent RNA
polymerase and accessory proteins are
bound to genomic RNA at regular intervals
b. Resistant to RNase
c. RNA polymerase only copies RNA when it is
in the nucleocapsid
and not when they are naked RNA
d. Nucleoproteins are single-stranded RNA
binding proteins
-Keep RNA single stranded
-Prevent base pairing between
template and product
-RNA synthesis takes place in nucleocapsids in the
cytoplasm or nucleus
IV. Double
-Virions
must contain and RNA polymerase
-Incoming RNA is
not infectious and cannot be copied or translated by
cellular
proteins
-Most are enveloped
-RNA synthesis takes place in subviral particles in the
cytoplasm
V. Examples of
RNA viruses
Orthomyxoviruses
A. Genus
a. Influenza A virus
b. Influenza B virus
c. Influenza C virus
d. Thogotovirus (Thogoto virus)
e. Isavirus (Infectious salmon anemia virus)
B. Example: Influenza
A virus
a. Genome Organization
i.
(-) strand
segmented RNA genome
ii.
Eight segments
encoding 10 proteins
b. Enveloped
c. Functions of
viral proteins
1. Hemagglutinin
1. Encoded by RNA segment 4
2. Membrane glycoprotein
3. Trimer of disulfide linked HA1 and HA2 molecules
4. HA0 is cleaved at basic residues by proteases located
in the trans golgi into HA1 and HA2 subunits
5. Mediate binding of the virion to host cells by
binding sialic acids attached to galactose with an a(2,6) linkage (human strains) or an a(2,3) linkage (avian strains)
6. The fusion peptide of HA (n-terminal regions of HA2)
mediates fusion of viral and endosomal membranes by undergoing a conformational
change at pH 5
7. Its cytolasmic domain binds the M1 protein which is
attached to viral RNA during assembly
8. Mutates rapidly
9. Influenza vaccines strive to produce antibodies
against Hemagglutinin to neutralize the virus by inhibiting cellular attachment
ii.
Neuramindase
1. Encoded by RNA segment 6
2. Membrane glycoprotein
3. Cleaves glycoside linkages of sialic acid
a. Inhibits aggregation of HA which are modified with
sialic acid in the golgi apparatus
b. Inhibits binding of HA to sialic acid residues on
host cells as virions are released from infected cells
4. Its cytomplasmic domain binds the M1 protein which is
attached to viral RNA during assembly
5. Mutates rapidly
6. Influenza vaccines strive to produce antibodies
against NA
iii.
Nucleoprotein
1. Encoded by RNA segment 5
2. Regulates the switch from viral mRNA to full length
(+) strand synthesis by binding to the nacent (+) strand and blocks poly A
addition
3. Contains a nuclear localization signal which allows
its import into the host nucleus by cellular import proteins
4. Facilitate nuclear localization of viral RNA after it
is released into the cytoplasm
5. M1 protein binds to both NP and cytoplasmic face of
HA and NA to guide vRNPs to the plasma membrane for assembly
iv.
Non-structural
protein
1. Encoded by RNA segment 8
2. Inhibits cellular mRNA polyadenylation
3. Inhibits splicing of cellular pre-mRNAs
4. Has a N-terminal RNA binding domain
5. Has a C-terminal domain that binds Cpsf and PabII
involved in polyadenylation
6. Cytoplasmic accumulation of mRNA may facilitate
cap-snatching
7. Sequesters dsRNA that could bind and activate Pkr
(Pkr phosphorylates eIF2a which shuts down translation)
8. Inhibits IFN synthesis
v.
Matrix Protein
(M1)
1. Encoded by RNA segment 7
2. Bind RNP and cytoplasmic tails of HA and NA
3. May undergo a conformational change before membrane
fusion facilitated by a low pH created by M2 allowing it to release vRNPs so they
can travel to the nucleus
4. It’s release is inhbited by the drug amantadine
5. By binding to genomic RNPs in the nucleus
a. Inhibits RNA synthesis
b. Promotes nuclear export of RNA
6. Has an intrinsic propensity to associate with
the plasma membrane
vi.
NEP
1. Encoded on RNA segment 8 by splicing
2. Contains a C-terminal M1 protein binding domain
3. Contains a N-terminal nuclear export signal
4. Directs export of vRNPs out of the nucleus
vii.
Ion Channel (M2)
1. Encoded on RNA segment 7 by splicing
2. The 5’ splice site for its mRNA is suboptimal and
requires binding of cellular protein sf2 to a enhancer
in the 3’ exon of M1 mRNA. This
interaction requires binding of P proteins with the 5’ end of M1 mRNA
3. Forms a homotetramer in the virion envelope
4. Activated by the low pH of the endosome before
membrane fusion occurs
5. Allows protons to enter the virion particle which may
cause a conformational change in M1 that causes its dissociation from vRNPs
6. Inhibited by amantadine
7. Increases the pH of normally acidid compartments of
the trans-golgi keeping HA in a fusion-incompetent state.
viii.
PA
1. Encoded on RNA segment 2
2. Part of the polymerase complex
3. Important for replication but how its initiation
properties are regulated is uncertain
ix.
PB1
1. Encoded on RNA segment 1
2. Part of the polymerase complex
3. Binds to the 5’ terminal sequence of genomic RNA
4. Binds to a 3’ terminal sequence of genomic RNA
activating its ability to cleave capped RNA
5. Cleaves capped cellular RNA
6. Catalyzes each nucleotide addition
7. Binds to a specific 5’ sequence on viral mRNA
inhibiting it’s cap cleavage
x.
PB2
1. Encoded on RNA segment 3
2. Part of the polymerase complex
3. Binding of PB1 to its RNA sequence induces a
conformational change allowing it to bind the 5’cap of cellular RNA
d. Single-cell reproductive Cycle
a. HA binds a sialic acid-containing cellular surface
protein or lipid
b. The virion enters the cell via receptor-mediated
endocytosis
c. Acidification of the endosome induces a
conformational change in M2 promoting proton influx
d. M1 undergoes a conformational change facilitating its
release from viral RNPs
e. HA undergoes a conformational change that exposes its
fusion peptide, facilitating viral and endosomal membrane fusion
f.
Viral
nucleocapsids are released into the cytoplasm
g. Viral nucleocapsids containing the segmented genome,
NP and Polymerase proteins are transported to the nucleus via nuclear
localization signals on NP
h. The (-) RNA genome is used to synthesize viral mRNA
via the viral polymerase complex which uses 5’ caps of cellular RNA as a
template
i.
Viral mRNAs
(spliced and unspliced) are transported to the cytoplasm by normal cellular
mechanisms through the nuclear pore complex
j.
Viral mRNAs of
HA, NA and M2 are translated on ribosomes on the ER and enter the secretory pathway where they
are modified by glycosylation.
k. All other mRNAs are encoded on ribosomes in the
cytoplasm
l.
P proteins and
NP are transported to the nucleus to facilitate synthesis of (+) strand RNA and
genomic RNA
m. M1, NEP and
NS1 proteins are transported to the nucleus and shut down viral mRNA production
and facilitate nuclear export of progeny nucleocapsids to the cytoplasm.
n. M1 association with NA and HA in the plasma membrane
facilitates virion assembly
o. The virion buds of from the host cell
p. NA cleaves sialic acids to prevent HA binding during
budding
e. Disease: Influenza
a. Symptoms
i.
Acute febrile
respiratory tract infection
ii.
Rapid onset of
fever, malaise, sore throat, cough
b. Transmission
i.
Inhalation of
small aerosol droplets
ii.
Most common in
winter when people are indoors
c. Mechanism
i.
Infects upper
and lower respiratory tract
ii.
Systemic
symptoms caused by a cytokine response
iii.
Neutralizing
antibodies to HA and NA are produced
iv.
Recovery depends
on a cell-mediated and interferon response
v.
May get a
secondary bacterial infection due to loss of natural epithelial borders
d. Treatment Strategies
1.
Killed vaccine containing antigens of two influenza
A viruses (H1N1 and H3N2) and a influenza B
virus
2.
Live attenuated influenza A and influenza B
vaccine (FluMist)
3.
Antiviral drugs
1. Amantadine
-Inhibits
the M2 ion channel
-Results in
retention of M1 to vRNPs
2. Rimantadine
3. Zanamivir
-inhibits NA
-delivered
via inhalation
4. Oseltamivir
-inhibits NA
-given
orally
e. Detection Methods
1.
Cell culture of nasal swab
2.
Immunofluorescence
3.
Z-stat test
Picornaviruses
A. Genus
a. Enterovirus (poliovirus)
b. Rhinovirus (Human Rhinovirus A)
c. Cardiovirus (Encephalomyocarditis virus)
d. Aphthovirus (Foot and mouth disease virus)
e. Hepatovirus (Hepatitis A)
f.
Parechovirus
(Human Parechovirus)
g. Erbovirus (Equine rhinitis B virus)
h. Kobuvirus (Aichi virus)
i.
Teschovirus
(Porcine teschovirus 1)
B. Example:
Poliovirus
a. Genome Organization
i.
(+) Strand RNA
genome
ii.
Vpg protein is
covalently attached to the 5’ end
iii.
Its RNA is
uncapped and translated by IRES
b. Non-enveloped
c. Function of viral proteins
i.
VP1
1. Part of the 5s structural unit
2. Upon virion binding to the Pvr receptor at
temperatures higher than 33 degrees Celsius, the virion undergoes a receptor
mediated conformational change that exposes its hydrophobic N-terminus
3. Its N-terminus inserts into the host cell membrane
forming a pore through which viral RNA is released from the capsid into the
cytosol
ii.
VP2
1. Part of the 5s structural unit
2. Part of VP0 with VP4 until viral RNA has been
encapsidated ant VP0 is cleaved by 3CDpro
3. Myristoylated
iii.
VP3
1. Part of the 5s structural unit
iv.
VP4
1. Part of the 5s structural unit
2. Part of VP0 with VP2 until viral RNA has been
encapsidated ant VP0 is cleaved by 3CDpro
3. Myristolated N-terminus interacts with VP3
4. Missing from the capsid once it has undergone a
conformational change upon receptor binding
5. Required for an early stage of cell entry
v.
2Apro
1. A protease
2. Cleaves the polyprotein precursor between P1 (capsid)
and P23 (protease and RNA synthesis)
3. Cleaves eIF4GI to inhibit translation of cellular
mRNA. The c-terminus of eIF4G is still
needed for recruitment of the 40s ribosome to IRES
vi.
2B
1. Inhibits membrane transport from the ER to the plasma
membrane
2. Synthesis of 2BC induces membrane vesicles by
interacting with the CopII complex and may prevent fusion with golgi by stabilizing the CopII complex. 2BC, 2B and 3A can all inhibit membrane
transport from the ER but only 2BC results in the typical polio-induced
membrane vesicles being produced
vii.
2C
1. Helps bring RNA polymerase to membranes
2. Anchors viral RNA to membranes during replication
viii.
3AB
1. Helps bring RNA polymerase to membranes (Binds 3Dpol)
2. Anchors the viral protein primer (Vpg) for RNA
synthesis to cell membranes
ix.
3CD
1. Precursor of 3Cpro and 3Dpol
2. The 3C portion contains the RNA binding domain
3. During (-) strand synthesis, binds the 5’ cloverleaf
structure of (+) strand along with poly (rC) binding protein 2. Interacts with the poly A binding protein
(PAbp) producing a circular genome.
Cleaves membrand bound 3AB to produce Vpg and 3A. Binds to the internal cre site to facilitate
Vpg-pUpU synthesis which is then transferred to the 3’ end.
4. During (+) strand synthesis it binds to the
cloverleaf in the (+) strand to separate the two strands and cleaves 3AB into
Vpg and 3A and cleaves itself to form 3Cpro and 3Dpol
x.
3Cpro
1. Cleaves VP0, VP1, and VP3 from P1
2. Cleaves 2A, 2B, and 2C from P2
3. Cleaves 3AB and 3CD from P3
4. Cleaves Vpg and 3A from 3AB
5. Cleaves 3Cpro and 3Dpol from
3CD
6. Cleaves the Tbp subunit of TFIIIb inhibitin RNA
polymerase II
xi.
3Dpol
1. The RNA-dependent RNA polymerase
d. Single-cell reproductive cycle
i.
Virion binds to
cellular Pvr receptor stimulating rearrangement
ii.
The hydrophobic
N-terminus of VP1 is exposed and inserts into the plasma membrane of the host
cell creating a pore
iii.
Viral RNA is
released into the cell cytoplasm through the pore
iv.
The 5’ attached
Vpg protein is removed from viral RNA
v.
The 40s ribosome
associates with the IRES of the viral RNA via eIF4G, eIF4A, and eIF3
interactions and binds near or at the AUG start codon
vi.
RNA is
translated into a polyprotein that is cleaved by 2A and 3C
vii.
(+) strand RNA
is brought to cellular vesicle membranes to initiate (-) strand synthesis
1. 2BC, 2B and 3A all inhibit membrane transport from
the ER and 2BC induced typical polio-induced membrane vesicles by interacting
with CopII
2. 2C anchors viral RNA to membranes vesicles
3. 3AB is anchored to the vesicle and serves as a
precursor for Vpg and can also binds 3Dpol to recruit it to the
membrane
4. poly(rC)-binding protein 2 (PCbp2) and 3CDpro
bind the 5’cloverleaf in the (+) strand of RNA
5. 3CDpro cleaves 3AB into Vpg and 3A
6. 3CDpro binds to the poly A binding protein
to create a circular genome
7. The internal cre sequence binds 3CDpro 3Dpol
and Vpg
8. Vpg-pUpU is synthesized by 3Dpol using the cre AAACA
sequence as a template
9. The complex is transferred to the 3’end and 3Dpol
uses Vpg-pUpU as a template for (-) strand synthesis
viii.
(-) RNA strand
is used to produce more (+) strand RNA for translation and packaging into
virions
1. The replicative form (-) strand is separated from the
parent strand by 2C which binds to the cloverleaf in the (-) strand and PCbp2,
3CDpro, and 3AB which bind to the cloverleaf in the (+) strand
2. 3CDpro cleaves 3AB into Vpg and 3A
3. 3CDpro cleaves itself into 3Cpro and 3Dpol
4. VPg-pUpU is synthesized by 3Dpol using the 3’ end of
the (-) strand as a template
5. 3Dpol synthesizes the (+) strand
ix.
Virion is assembled by formation of 5S
structural units (VP0, VP1 and VP3) that assemble into 14S pentamers.
x.
14S
pentamers associate with (+) strand
viral RNA to form a 150S non-infectious virion stabilized by protein-protein
interactions and interactions mediated by myristate chains of VP0
xi.
An infectious
150S virion is formed by cleavage of VP0 into VP2 and VP4.
xii.
Release from
host cell by lysis (mediated by 3A induced increased in membrane permability)
or non-destructive apical release as seen in GI epithelial cells
e. Disease: Polio
i.
Symptoms
1. Paralysis
2. Enchephalitis and meningitis
3. Respiratory tract infections
4. Undifferentiated fever
5. Disease in immunodeficient patients
ii.
Transmission
1. Fecal-oral route
2. Polio is nearly eradicated
iii.
Mechanism
1. Enters oropharyngeal or intestinal mucosa and
replicates
2. Secretory IgA can prevent infection
3. Travels to the lymph note
4. Spread by Viremia to the brain and meninges
5. Virus shed in feces
iv.
Treatment
Strategies
1. Live oral or inactivated vaccines
v.
Detection
Reoviruses
A. Genus
a. Orthoreovirus (Mammalian orthoreovirus)
b. Orbivirus (Bluetongue Virus)
c. Rotavirus (Rotavirus A)
d. Coltivirus (
e. Aquareovirus (Aquareovirus A)
f.
Cypovirus
(Cypovirus 1)
g. Fijivirus (Fijivirus 1)
h. Phytoreovirus (Rice dwarf virus)
i.
Oryzavirus (Rice
ragged stunt virus)
B. Example: Mammalian
Orthoreovirus
a. Genome Organization
i.
Double stranded
RNA
ii.
10 dsRNA
segments
iii.
Viral RNA
polymerase and other enzymes needed for mRNA synthesis are packaged in the
particle
b. Non-enveloped and Icosahedral
c. Function of Viral Proteins
i.
l1
1. Makes up the dense core shell
ii.
l2
1. 5” capping enzyme
2. Guanylyltransferase
3. N-methyltransferase
iii.
l3
iv.
m1
1. Makes up the outer capsid
2. Mediates interaction of ISVP with membranes
3. Present in the virion as two cleaved fragements m1C
and m1N. Cleaved by lysosomal proteases. They function in membrane penetration
v.
m2
vi.
mNS and mNSC
vii.
s1 and s1s
1. Attaches to the cell receptor (sialic acids)
viii.
s2
1. Makes up the dense core shell
ix.
sNS
x.
s3
1. Makes up the outer capsid
2. Released by proteolysis in the ISVP
3. Sequesters dsRNA to inhibit Pkr activation
d. Single-cell reproductive cycle
i.
The virion
attaches to the host cell via s1 binding sialic acid on the host cell
ii.
The virion
enters by receptor-mediated endocytosis
iii.
Proteolysis in
the late endosome by lysosomal proteases of s3
and m1 creates the ISVP (infectious subviral particle)
iv.
m1N and m1C mediate membrane penetration
v.
The core is
formed by releases of 12 s1 and 600 m1 subunits
vi.
Synthesis of 10
capped viral mRNAs begins in the core
vii.
l2 facilitates 5’ capping of mRNA
which lacks a poly-A tail
viii.
mRNAs are
translated and associate with newly synthesized viral proteins to form RNase
sensitive subviral particles
ix.
mRNA also serves
as a template for (-) strand RNA synthesis leading to the production of
dsRNA containing RNase-insensitive
subviral particles
x.
mRNAs in
subviral particles facilitate further protein synthesis
xi.
Preformed
complexes of outer capsid proteins are added to subviral particles
xii.
Mature virus
particles exit the cell by lysis
e. Disease
i.
Symptoms
1. Mild upper respiratory tract disease
2. Gastroenteritis
3. Biliary atresia
ii.
Transmission
1. Fecal-Oral route
iii.
Mechanism
1. Ingestion of contaminated food or water
2. Large quantities of virions released in diarrhea
3. Immunity to infection depends on IgA in the gut
iv.
Treatment
Strategies
1. None
v.
Detection
Rhabdoviruses
A. Genus
a. Vesiculovirus (vesicular stomatitis virus
b. Lyssavirus (Rabies Virus)
c. Ephemerovirus (Bovine Ephemeral fever virus)
d. Novirhabdovirus (infectious hematopoietic necrosis
virus)
e. Cytorhabdovirus (Lettuce nectrotic yellows virus)
f.
Potato yellow
dwarf virus
B. Example:
Vesicular Stomatitis Virus
a. Genome Organization
i.
(-)Strand RNA
ii.
Non-segmented
b. Enveloped
c. Function of Viral Proteins
i.
N
(Nuceloprotein)
1. Complexes with P at a 1:1 molar ratio
2. Aggregates when synthesized alone
3. NP-Complexes suppress mRNA synthesis by binding to leader
RNA causing anti-termination
4. Additional N proteins bind to the (+) RNA as it
elongates and 7 A bases in the intergenic region. Prevents the
A bases from slipping back on genomic RNA and blocks reiterative coping
of the 7 U bases by L-P polymerase
5. As genomic RNA is synthesized, 10 N molecules and 5 P
molecules bind 90 nts of RNA to form oligomers facilitating ribonucleotide
formation
ii.
P (RNA
polymerase)
1. Forms a complex with L (L-P complex) to form an RNA
polymerase that synthesizes mRNA
2. Forms a complez with N to form a (L-PN) complex that
synthesizes viral genomic RNA
3. NP complexes suppress mRNA synthesis by binding to
leader RNA causing anti-termination
4. Forms oligomers with N proteins and genomic viral RNA
to form ribonucelotides
5. Bind the RNA genome in the virion
iii.
M (Matrix
Protein)
1. Binds to the internal domain of envelope glycoprotein
G and to Ribonucleoproteins (N-terminus) aiding in packaging of viral RNPs
2. Inhibits transcription by host RNA polymerase II
iv.
G (Glycoprotein)
1. Has a palmitate link
2. Associates with the matrix protein
v.
L (RNA
polymerase)
1. Carries out all reactions necessary for capping and
synthesis of viral mRNAs
2. Bind the RNA genome in the virion
vi.
Leader Sequence
1. inhibits both RNA polymerase II and III
2. enters the nucleus and may bind cellular proteins
d. Single-cell reproductive cycle
i.
Virion binds
cell receptor (?) can enters the cell via receptor-mediated endocytosis
ii.
The virion
membrane fuses with the endosome membrane
iii.
Viral
nucleocapsid is released into the cytoplasm
iv.
The (-) strand
is copied into five subgenomic mRNA
strands by L and P proteins
v.
N, P, L, and M
proteins are synthesized on ribosomes in the cytomplasm
vi.
G protein is
synthesized by ribosomes attached to the ER, enters the secretory pathway and
is transported to the plasma membrane
vii.
N, P, and L
proteins synthesize a full length (+) strand that serves as template for
genomic RNA synthesis
viii.
N and P-L
associate with genomic RNA
ix.
Matriz protein
binds N and facilitates virion assembly by also binding protein G at the plasma
membrane
x.
The virion buds
from the host cell basolaterally
e. Disease
i.
Symptoms
1. Flu-like Illness in humans
2. Acute infection producing vesicular lesions in
cattle, pigs, and horses
ii.
Transmission
iii.
Mechanism
1. CD4 T cells can accomplish non-cytolytic clearing of
the virus
iv.
Treatment
Strategies
1. Used as a viral vector in vaccine development to
deliver high titers by a mucosal route
v.
Detection
Togaviruses
A. Genus
a. Alphavirus (Sindbis Virus)
b. Rubivirus (Rubella Virus)
B. Example: Sindbis Virus
a. Genome Organization
i.
(+) strand RNA
ii.
Genomic RNA has
a 5’cap and 3’ poly-A tail
b. Enveloped
c. Function of Viral Proteins
i.
Capsid Proteins
ii.
E1 protein
1. Envelope protein
2. Forms a dimer with E2 in the ER
iii.
E2 protein
1. Envelope protein
2. Forms a dimer with E1 in the ER
3. Has a palmitate modification
iv.
E3 protein
1. Envelope protein
v.
6K
1. Envelope protein
vi.
nsP1
1. Part of (+) strand polymerase
vii.
nsP2
1. Part of (+) strand polymerase
2. Protease
viii.
nsP3
1. Part of (+) strand polymerase
ix.
nsP4
1. Part of (+) strand polymerase
2. Encodes the RNA-dependent RNA polymerase
x.
P123 and nsP4
1. (-) strand RNA polymerase
d. Single-cell reproductive cycle
i.
Virion binds to
a high affininty laminin receptor on a host cell
ii.
Virion enters
cell via receptor mediated endocytosis
iii.
Upon
acidification of the endosome, viral RNA is released into the cytoplasm
iv.
Viral (+) RNA is
translated into the P1234 polyprotein and cleaved at various sites by nsP2 to
form different RNA polymerases
v.
P123 and nsP4
copies viral RNA into (-) strand RNA
vi.
nsP1, nsP2,
nsP3, and nsP4 copy viral RNA into (+) strand RNA and subgenomic mRNA
vii.
Subgenomic mRNA
is translated on free ribosome in the cytoplasm until the Capsid protein is
released by proteolytic cleavage
viii.
Cleavage of the
capsid protein exposes a hydrophobic N-terminal domain of PE2 and the ribosome
associates with the ER
ix.
PE2, K6, and E1
proteins enter the secretory pathway and are brought to the plasma membrane
x.
The capsid
protein associates with viral (+) full length RNA and brings it to the plasma
membrane by associating with viral glycoproteins
xi.
The virion buds
from the host cell
e. Disease
i.
Symptoms
1. Subclinical
ii.
Transmission
1. Trasmitted by the bite of a Aedes mosquito
iii.
Mechanism
1. Bite by Aedes Mosquito
2. Virus enters bloodstream (viremia)
3. Antibodies in blood limit spread by viremia (IgM and
IgG)
4. Cell-mediated immunity is important to resolve
infection
iv.
Treatment
Strategies
1. No antiviral drugs
2. Rubella (Rubivirus) has a live, attenuated vaccine
v.
Diagnosis