Lec 8 Orthomyxovirus Flashcards
Orthomyxoviridae (Influenza)
Orthomyxovirus
Enveloped, single stranded RNA, NEGATIVE sense (Complement to mRNA),
SEGMENTED genome (8 segements each coding for DIFFERENT GENES). All 8 are necessary for virulence. Ramifications: If 2 different influenza viruses infect one cell, they can reassert their genes to create new strains!
The only RNA virus to replicate in HOST NUCLEUS and to REQUIRE HOST mRNA synthesis to replicate.
OTHER viruses REPLICATE in the CYTOPLASM, don’t need nucleus. Orthomyxovirus has problems getting to nucleus but there are pros.
H1N1 outbreak in Mexico in 2009 towards the world.
2nd wave in 2010.
Has characteristics of pandemic infections. Kills immunocompromised individuals (young and old)
Orthomyxoviridae Influenza Disease
Symptoms include high fever, sore throat, cough, headache, muscular pain.
Can be FATAL in the elderly, infants and chronically ill often as a result of SECONDARY BACTERIAL INFECTIONS. Influenza depresses immune system even more
Emerging avian influenza virus strains threaten domestic fowl and may adapt and become a source leading to a human pandemic. Virus can change immunogenic proteins through gene mutations in different animals.
B/c this virus is adaptive you need a flu shot every year.
Orthomyxoviridae Influenza virus infection
Causes epidemic disease, first account of symptoms in 412 BC by Hippocrates.
First believed to be bacterial disease
in 1920 identified virus etiology
virus first isolated in 1933 by Smith et al.
Epidemics frequent and regular severe pandemics.
Most appear to come from China and spread through Russia to the rest of the world
Pandemics over the last hundred years: H7N9 epidemic in China 2013 Pandemic (H1N1) 2009 Influenza, 2009–2010 Hong Kong influenza, 1968–69 Asian influenza, 1957–58
SPANISH INFLUENZA MOST SEVERE PANDEMIC 1918-1919 killed more people (20-50 Million) than 1st world war (20 million)
Orthomyxoviridae Virus Morphology
Pleomorphic enveloped virus, in culture typically ovoid, 80-120 nm
Enveloped: much less structured than non enveloped complex, icosahedral shape
Pleomorphic: does NOT look symmetric, irregular eg. The virus all look different, ovoid or irregular
Envelope, host plasma membrane lipid
Matrix proteins are located underneath membrane.
Membrane has structural proteins and
3 long -ssRNA segments, 5 shorter ones, all key shaped and have RNA pol complex made of PA PB1 and PB2 proteins!
RNA polymerase does NOT HAVE PROOFREADING: 1 mistake / 1000 NTP: mutations allows virus to adapt to different host and make different proteins VERY rapidly!
2 antigenic proteins HA (hemaglutinin) and NA (neuraminidase) and
M2 protein (ion channel allows maintaining pH in virus)
NS2 protein in the centre of the virus.
NP: Nucleocapsid protein coats RNA.
Orthomyxoviridae Spike proteins
2 spike proteins HA & NA in ~4.5/1 ratio (HA:NA)
Immunogenic part of the virus, examined by immune system.
HA (hemagglutinin) aggregates to form TRIMER, split into. This protein causes agglutination of RBCs
HA1 and HA2 subunits folded and linked together by disulfide bonds. CONTIGUOUS in one part, when cleaved becomes a fusion peptide to fuse virus to host cell.
HA2 is fusion domain pH DEPENDENT(low pH needed)
HA1 binds SIALIC ACID in host.
NA (Neuraminidase), forms a club like shape and CLEAVES SIALIC ACID. Advantage is that when the virus buds out of cell, it does not remain bound and escapes faster.
Orthomyxoviridae (Influenza) Types A B C
Influenza A is the cause of MAJOR EPIDEMICS, infects humans, swine, birds, horses etc.
Variable HA & NA proteins, serotype variable
HA (1-16 subtypes) NA (1-9 subtypes)
**Reassortment happens mostly in A
limited number of subtypes have established species-specific lineages in Humans(H1N1,H2N2,H3N2).
Influenza B, only infects humans and doesn’t show same variability in HA & NA
Influenza C, distinct morphology, only a single membrane protein, 7 rather than 8 segments HEP instead of HA and NA, infects Humans & swine
Orthomyxoviridae Matrix Protein M1 and M2
Matrix Protein, (M1 & M2),
Gene segment 7 codes for M1 but produces low levels of M2 by alternate splicing
- M1 is typical matrix protein found between MEMBRANE and CAPSID structure,
- transports newly made RNP from NUCLEUS TO MEMBRANE for assembly, associates with RNP and
- binds membrane proteins including M2 tightly
M2 is found in membrane in low copy number, associates with M1, acts as an ION CHANNEL TO DROP pH and allow RELEASE of RNP core from phagolysosome surface
Helical Nucleocapsid of Influenza virus (1 segment)
Influenze A virus
RIBONUCLEOPROTEIN CORE COMPLEX: Composed of 8 separate segments of -ve sense RNA,
each associated in a core structure made up of 4 different proteins.
Orange balls: bind RNA.
PA PB1 PB2 are polymerase complex.
Must first e transcribed into +mRNA and serves as template for - strand.
Orthomyxoviridae Ribonucleoprotein Core Complex (RNP)
NP (nucleocapsid protein), forms shell around RNA, lose association with RNA leaves RNA susceptible to RNAase activity, binds both gRNA and cRNA, Full length. NP does not bind complementary strand. If RNA is not protected by NP it becomes sensitive to RNAses
RNA has encapsidation sequence in 5’ end absent in mRNA
PB1, motifs typical of RNAdRNAp, basic protein, binds 5’ and 3’ region of gRNA
PB2, also a basic protein, cap-binding protein of host mRNAs. PB2 STEALS host caps and uses them to start vRNA transcription!
PA, acidic protein, Polymerase subunit: Protease activity, RdRp subunit.
Orthomyxoviridae Attachment, Penetration, Uncoating
- Attachment involves HA binding to sialic acid of glycoproteins and lipids
low affinity requires multiple binding sites for efficient binding and entry. - Penetration into endosome via RECEPTOR MEDIATED ENDOCYTOSIS
- pH mediated membrane fusion by HA protein
- M2 acts as proton transporter to drop pH at inverted membrane and allow release of RNP from M1 protein
RNP enters nucleus via NUCLEAR PORE COMPLEX
Orthomyxoviridae Protein list
EE AT ECV MT ME LE
EE: early endosome AT: Actin dependent way ECV: Endocyctic carrier vesicle MT: Microtubule ME: Maturing endosome LE: Late endosome
Virus enters w/ clathrin coated pit into early endosome, attaches to microtubule, uses dye nine and kinesin to MTOC, can use endocytic carrier vesicle or early endosome to move! Early endosome matures and decreases endosome pH AND virus so virus fuses to late endosome membrane to be released into cell and targets nucleus for replication.
Orthomyxoviridae Penetration through cellular membranes
Fusion proteins undergo major conformational changes that lead to fusion
Activated by low pH or receptor binding
For folded HA protein to carry function, decreased pH and host protease changes HA conformation to extend it and expose the sialic acid binding domain, which attaches to endocytic vesicle surface.
Endocytic Trimers aggregate to cause another conformation change in HA protein, allows HA to bend over and pull membrane together to cause invagination. Once host membrane and viral membranes are close together a channel forms and releases nucleocapsid core into cytoplasm.
Orthomyxoviridae Transcription
Takes place in nucleus, requires mRNA synthesis by host, (blocked in cells treated with actinomycin D or a-amanitin)
PB1 cleaves mRNA and binds A from AUG to viral RNA’s U to serve as primer for mRNA b/c this A is bound to cap. PB1 complex rolls vv genome to multi U portion, transcribed to make viral poly A tail until termination ends to make a full viral mRNA! Until proteins get back into nucleus, genome transcription cannot occur!
Caps removed from host mRNA serve as primers for viral mRNA synthesis
virus can’t make its own caps
Viral mRNA synthesis requires vRNA templates, NP, PB1, PB2, and PA proteins, and host mRNA
Transport of influenza virus RNAs and Proteins between nucleus and cytoplasm
Occurs in nucleus, needs host mRNA and vRNA synthesis
For 1 segment, its delivered from late endosome to nucleus, transcribed to make 5’cap and poly A mRNA, transported back out into cytoplasm for translation, viral proteins transported back into cytoplasm to form around ANTIGENOME, ** replication of vRNA into genome w/ M1 and NS2 proteins to bind and form new segment structure.
Nucleocapsids are exported from the nucleus in a complex with M1 and NS2
NS2 has a nuclear export signal and can bind M protein it thus signals the host export machinery to transport the RNP to the nucleopore and into the cytoplasm
Orthomyxoviridae RNA Replication
Last few steps
Switch to replication from transcription requires viral protein synthesis.
Replication requires a move from cap initiated synthesis and poly A addition to full copies of genomic RNA to complementary RNA, and cRNA to gRNA
NS proteins especially NS1 involved, as is NP, in switch, issue also as to what makes up polymerase complex
Regulation of mRNA and protein synthesis, NS & NP dominate early, M & HA are late proteins
In terms of mRNA, NS and NP dominate early and M and HA dominate late.
Occurs with PB1 attaching and peind used as primer for cap site, now starting with a coated RNA (with NP)
PB1 takes away NP and transcribes genome to the end. Create + anti genome from - genome, then transcribe anti genome back to genome to be encapsidated.
