Lec 4 Flavivirus Flashcards
Flavivirus
Pestivirus and Hepacvirus have IRES to orient ribosomes
1 large ORF
Polyprotein made is self processed by proteases found in genome
Don’t memorize everything.
Capsid proteins (C) E protein chaperone E is responsible for binding and fusion,
Methyltransferase encodes RdRp
Protein that cleaves polyprotein is Protein3, which binds w/ MS3 (cofactor) to activate
Flavivirus Viral proteins
+ssRNA Replication complex
NS1-translocated to ER lumen, may function in RNA synthesis
NS2A-localized in RNA replication complex, binds NS3 and NS5 as well as non-coding regions of RNA.
Believed to cross ER membrane more than once?
NS2B-Spans membrane many times, co-factor in NS3 protease activity.
NS3-serine protease responsible for processing of viral cytoplasmic proteins
Polyprotein is translated in ER membrane, then cleaved. Depending on which side of the membrane the polyprotein is on, the protein goes on the cytoplasm/lumen
Flavivirus Replication Viral Proteins
NS4A/4B and 5
NS4A/4B-Speculated to be involved in RNA replication, remains membrane associated
NS5-Largest of the NS proteins, serves as the RNAdRNAp, possessing the typical motif found with viral RNAp. Also gives rise to methylated-cap (5’) of viral RNA to make it look like a host mRNA!
Protein synthesis occurs along ER for membrane protein processing
Nascent C protein has a C-terminal signal sequence that translocates PrM to the lumen of the ER
Similar sequences at C-terminal of PrM translocates E, and one at the end of E translocates NS1 to the lumen.
Signal peptidases cleave these proteins
NS2B-NS3 COMPLEX is the protease that cleaves C from PrM, and cleaves most Non Structural proteins
Membrane topology of the flavivirus structural protein
N term. is in cytoplasm, protein inserted into membrane (many Membrane spanning domains)
Most PrM is on ER membrane
Most E on ER lumen
Most NS1 is on ER lumen too
NS3 and 5 are on cytoplasmic side.
Capsid protein that coats genome is on cytoplasmic side
Signal peptidases and Purin cleave on the ER lumen side.
THEREFORE virus buds from the ER membrane out
Capsid protein form capsid, membrane proteins on ER side of membrane form membrane during budding process.
Nonstructural proteins stay in the ER membrane. When its in the lumen the bud is a separate, immature virion
Flavivirus Replication Cycle
1.
- At ER membrane, RNA is uncoated and attaches to ER ribosome to get translated. Polyprotein is inserted into ER membrane.
- RNA is replicated on cytoplasmic site of ER membrane, and virus coats this RNA to bud into the ER.
- Fully formed virus in the ER lumen escapes.
Flavivirus Replication mech
vRNA acts as template for cRNA synthesis, and cRNA then copied to make more vRNA (-sense)
NS3 and NS5 appear to be polymerase complex for viral genome
RNA replication involves both RF (replicative forms, duplex RNA),
RF form produce low levels of complementary RNA, and
RI (replicative intermediates, contain DS-regions and nascent SS-regions) producing high levels of vRNA
vRNA 10 fold greater than cRNA
The cell is able to replicate vRNA b/c cell does not want to spend time making - ssRNA, make as less as possible to make vRNA
Repiication begins de novo with out any peptide or RNA primer and may involve the cyclization sequence
Flavivirus Temporal Regulation
- Polyprotein
- NS5 and other proteins transcribe +ssRNA into viral template
- Come back here
Flavivirus Viral Assembly
Viral assembly takes place in the cytoplasm, internal membranes sites of viral envelope production and virus budding, however low level of budding may occur at plasma membrane (depends on the flavivirus genera, uncommon)
Cytoplasmic tail of PrM interacts with C in docking process. Exposed to the side where capsid proteins are in, so nucleocapsids move to where viral proteins are put into membrane.
PrM and E proteins complex to prevent adhesion and fusion of virus to dead cell and internal membranes! This is step 7.
PrM and E dissociate after virus release
Flavivirus Virion released through exocytosis pathway
Virions are in vesicles which fuse with plasma membrane
PrME blocks virus from fusing to internal membrane, and from infecting dead cells until the virus matures to become infectious.
In the trans-golgi Furin- mediated cleavage of prM leads to M and generates mature infectious particles
Flavivirus Distinctive Characteristics
Misc.
Flaviviruses cause severe disease in humans:
ZIKA, Yellow Fever, Japanese encephalitis, Dengue fever, West Nile Virus and Hepatitis C
Many Flaviviruses are transmitted from host to host via mosquitoes or ticks. Hot spots
But not Hepatitis C virus!
Birds may be intermediate hosts. Mosquito bite the birds to get virus.
West Nile came from Africa, to NY d.t. birds.
Flavivirus Distinctive Characteristics
Mechanisms
Viral proteins are synthesized as a single polypeptide that is cleaved by viral and host cell proteases
Pestiviruses and Hepaciviruses (genera) have an internal ribosome entry site for protein synthesis initiation. But others have a vRNA with a 5’ Cap.
RNA synthesis takes place on cytoplasmic membranes
Virions are assembled by budding at endoplasmic reticulum and are released by exocytosis
ZIKA virus transmission
a Flavivirus!
ZIKV can be transmitted sexually and during pregnancy from a mother to her fetus.
In addition to microcephaly, ZIKV causes miscarriage.
ZIKV persists in whole blood for close to 2 months.
Dengue viral infections enhance ZIKV infections.
Anti-ZIKV antibodies in domestic animals suggest
that ZIKV can infect domestic animals.
Flaviviridae Fevers
Yellow fever virus is the type member for the flaviviruses Many victims suffer from hepatitis and this tinges the skin yellow.
West nile virus infections not a major health treat but can cause neurologic problems and is occasionally fatal
(three stages
1) high fever and muscle pain, headaches, and severe vomiting;
2) a quiescent state in which the fever disipates and the patient appears to be in remission
3) repeat of original symptoms in more severe form resulting in Multiorgan failure with a mortality between 20-50 %.
In insects DENGUE infects the midgut and then spreads to the body cavity and organs
Hepacivirus
What kind of virus?
Hepacivirus
Hepatitis C Virus (HCV), originally called non-A non-B hepatitis now identified as a Flavivirus, common form of hepatitis spread in blood products, humans only known host, disease ranges from mild carrier state or liver cirrhosis to inducing liver cancer (Hepatocellular carcinoma)
Hepatitis C virus transmitted by blood transmission, sharing of contaminated needles of intravenous drug abusers and by sexual contact but not by an arthropod host.
Flavivirus Virus Structure
Enveloped icosahedral virus (37-50 nm)
Envelope contains 2 glycoproteins E and M (starts as pre-M), both having membrane spanning regions
Capsid (spherical about 30 nm) is made of a single capsid protein of 12 kd (Capsid Protein)
