BIOC20 - Lecture 8 - Negative Strand RNA Viruses - PDF

Lecture 8 Negative strand RNA viruses Filoviruses Structure Disease and spread Genome, replication and translation RNA editing and protein function Virus entry and exit Latent infections Influenza viruses (introduction) Types...

Lecture 8 Negative strand RNA viruses Filoviruses Structure Disease and spread Genome, replication and translation RNA editing and protein function Virus entry and exit Latent infections Influenza viruses (introduction) Types Disease and spread Genome and proteins 1  Filoviruses - Structure Filoviridae from Latin filum (filament or thread like) Filamentous, enveloped particles Diameter 80 nm, length of 800 nm or more Helical nucleocapsids with negative sense ssRNA genome Genome size ~15-19 kb 2 EM image of Marburg virion Model of Marburg virus Schematic of Ebola virus  Filoviruses - Structure Filoviridae from Latin filum (filament or thread like) 3  Filoviruses – Disease and Spread First outbreak of Marburg virus was in Marburg and Frankfurt, Germany in 1967 Laboratory workers processing monkey tissues - 32 cases, 7 died (22% mortality) A similar virus emerged (Ebolavirus, EBOV) in 1976 by two independent epidemics in Zaire (now Democratic Republic of Congo) and Sudan Distinct EBOV species: Sudan 4 ebolavirus (SEBOV) and Zaire  Filoviruses – Disease and Spread Marburg and Ebola viruses are sporadically re-emerging viruses that cause severe, often fatal, disease ZEBOV & SEBOV commonly/continuously re-emerge in Africa 1994 gave rise to 3rd EBOV species from Cote d’Ivoire  Cote d’Ivoire ebolavirus (CIEBOV or Tai Forest) Experimental monkeys shipped from Philippines to Reston, Virginia (USA) in 1990 – rare and non-pathogenic in humans  Reston ebolavirus (REBOV) Bundibugyo Ebola virus (BEBOV) discovered in Uganda (2007) More recent EBOV outbreak in 2014-15 was the largest outbreak in history: Originated in Guinea 5  Filoviruses – Disease and Spread Despite public perception, Ebolavirus is unlikely to spread disease with large numbers of casualties No vaccine is available for SEBOV ZEBOV vaccine has a global stockpile stored in Switzerland  can be shipped within 48 hours for emergency response. 6  Filoviruses – Disease and Spread Two genera: Marburgvirus and Ebolavirus Ebolavirus species are named after their original site of discovery: Bundibugyo, Cote d’Ivoire, Reston (not associated with human disease), Sudan and Zaire There is a single Marburgvirus species: Lake Victoria Marburgvirus These viruses are probably transmitted to primates from fruit bats (zoonotic spread) Infections with Marburg and Ebola viruses cause severe haemorrhagic fever  major symptoms are fever, haemorrhages, liver dysfunction, intravascular coagulation (blood clotting), cytokine release, vascular permeability and shock. Lethality can be high (up to 90%)! Recent advances: a highly effective Canadian-made vaccine and others! 7 ➔  Filoviruses – Disease and Spread Spread of filovirus infections among humans is limited to close contacts Aerosolized human-human spread is low risk Person-person transmission is mediated by physical contact with secretions/excretions e.g. blood, feces, vomit, urine, semen Close family members and medical staff are the most at-risk Epidemics usually are self-limiting, as virus transmission period is transient and generally ineffective Another example of zoonotic spread 8  Filoviruses – Genome and Proteins Linear, negative sense, single-stranded RNA genome (‘-’ ssRNA) Genome lengths ~15-19 kb Seven genes, transcribed in series from 3’ end of genome by viral RNA polymerase Most genes produce a single mRNA and a single protein Orientation depicted as 3’ to 5’ indicates Fig. 16.3, Ebola virus genom ‘-’ sense genome 9  Filoviruses – Genome and Proteins Filovirus genomes contain seven genes in a conserved order Each gene codes for a single proteins, except for GP  gets cleaved into GP1 and GP2 or sGP (secreted GP) All genes flanked by conserved sequences that signal transcription termination, polyadenylation and reinitiation Individual transcripts made for each viral protein Fig. 16.3, Ebola virus genom 10  Filoviruses – Genome and Proteins Most proteins are packaged in virion: Nucleocapsid protein (NP) RNA polymerase cofactor (VP35) Matrix protein (VP 40) Envelope glycoproteins (GP, cleaved into GP1, GP2 or sGP) Minor nucleocapsid protein (VP30) Fig. 16.2 Membrane protein (VP24) RNA polymerase (L) Ebola makes additional secreted glycoproteins (sGP, delta-peptide) 11 Marburg virus genome Ebola virus genome  Filoviruses – Genome and Proteins Proteins names are based on function, glycosylation status or molecular weight. The four red highlighted proteins are associated with genomic RNA and / or nucleocapsid. The blue highlighted proteins are associated with the viral envelope. 12  Filoviruses – Replication Transcription and replication occurs in cytoplasm similar to other ‘-’ sense RNA viruses e.g. Paramyxovirus The template for mRNA synthesis is the ‘-’ sense RNA genome 3’ leader contains promoter for viral RNA polymerase and packaging signal for assembly of nucleocapsids Fig. 16.3, Ebola virus genome Fig. 15.8 (Paramyxovirus transcription and replication, modified 13  Filoviruses – Replication 1. Viral RNA polymerase Fig. 16.3, Ebola virus genom begins transcription at 3’ terminal of the genome (high NP levels) 1 2. When little or no free NP protein (during initial infection) is present  2 RNA polymerase NP (low NP levels) transcribes a short VP35 sequence, then VP40 GP/sGP terminates to release a VP30 free leader RNA and VP24 then scans for nearby mRNA start site and re- 14 initiates transcription at Fig. 15.8, modified  Filoviruses – Replication (low NP levels) 3. L protein adds 4 methylated 5’ cap and (high NP levels) polyA tail, like cellular mRNAs 3 (low NP levels) 4. Once sufficient NP NP made, genome VP35 VP40 replication can begin GP/sGP VP30 VP24 15 Fig. 15.8, modified  Filoviruses – Replication Genome replication occurs via synthesis of antigenome RNA (low NP levels)  serves as template for synthesis of new negative- strand RNA genomes: Viral RNA polymerase (high NP levels) changes its mode of synthesis: no longer recognizes termination-polyA- re-initiation sites (low NP levels) NP RNA pol extends the growing VP35 VP40 chain to end of template GP/sGP without stopping VP30 VP24 Genome RNAs are packaged into nucleocapsids during replication  replication only 16 Fig. 15.8, modified  Filoviruses – Replication The template for both mRNA synthesis and genome replication is the ‘-’ RNA genome The cytoplasm of infected cells contain inclusion bodies which contain viral nucleocapsids Inclusion bodies: Aggregates of (viral) proteins / nucleic acids within the host cell mCherry: red fluorescent protein fused with L protein EBOV inclusion body formation time course 488: indicates a green fluorescent dye (Alexa Flour 488 17 analysis  Filoviruses – RNA Editing Ebola virus uses RNA editing to make two glycoproteins from the same gene: sGP (~80%): a shorter protein that does not include the transmembrane domain at the C terminus and is secreted! Also yield delta-peptide upon proteolytic cleavage. GP (20%): RNA polymerase “stutters” and adds an extra ‘A’ residue to N-terminal change the reading frame to yield full length GP. C-terminal Notice the Thr to Asn change Fig. 16.6 18  Filoviruses – RNA Editing The editing site contains a stretch of 7 ‘U’s  transcribed into ‘A’s in ‘+’ sense mRNA Occasionally the viral RNA polymerase will ‘stutter’ over this stretch of ‘U’s and add an additional ‘A’  changes Thr to Asn and changes the reading frame downstream. In some cases, it may be 6 or 9 ‘A’s producing a smaller sGP (ssGP) No delta peptide produced 19  Filoviruses – RNA Editing Editing of Ebolavirus gene product can produce sGP (most common), GP, ssGP (least common) 20  Filoviruses – Attachment and Entry GP is synthesized as a precursor protein, inserted into the lumen of ER: An N-terminal signal sequence is cleaved off during insertion in ER Protein undergoes extensive glycosylation in ER and Golgi  transport to plasma membrane Cellular furin protease then cleaves GP into the ectodomain GP1 (N-terminal) and transmembrane GP2 (C-terminal) GP1 and GP2 held together by a disulfide Fig. 16.5 bridge between two cysteine residues (cysteine bridge) 21  Filoviruses – Attachment and Entry Filovirus GP mediates attachment and entry (by fusion) GP mediates binding to multiple cellular receptors Asialoglycoprotein receptor: Liver-specific, binds to and internalizes glycoproteins that lack terminal sialic acid Folate receptor-α: binds to folic acid Integrins: cell-surface proteins that interact with extracellular adhesion proteins and initiate intracellular signalling DC-SIGN: dentritic cell-specific intracellular adhesion molecule-grabbing nonintegrin – type II transmembrane proteins that bind mannose and involved in interaction with T cells Most binding experiments have been performed using replication-deficient pseudotypes (recombinant / pseudo virus) 22  Filoviruses – Attachment and Entry Filovirus pseudotypes have GP incorporated into the envelopes of unrelated recombinant viruses  can be used to study viral proteins e.g. GP for attachment and entry. This technique could be a bit problematic to study attachment and entry. Why? Generating pseudotypes requires various vectors that are co-transfected: This image shows SARS- CoV2 pseudovirus. Which vector would you change for filovirus GP? 23  Filoviruses – Attachment and Entry Virus is taken up via macropinocytosis and using its fusion peptide (N- terminal of GP2) it fuses within vesicles to enter cells (probably a low pH trigger) 24  Filoviruses – Protein Function Proteins names are based on function, glycosylation status or molecular weight. The four red highlighted proteins are associated with genomic RNA and / or nucleocapsid. The blue highlighted proteins are associated with the viral envelope. 25  Filoviruses – Protein Function sGP is released from infected cells and is found in serum of infected patients  can be used as a biomarker and as a potential vaccine / antiviral target. Function is not entirely clear: Considered non-structural but may substitute as a structural protein by forming a complex with GP2 Some pseudotyping data shows that it may limit GP cytotoxicity  more efficient replication and infectivity (?, speculation) Acts as a soluble factor that targets elements of the host defence system e.g. binding to antibodies and contribute 26 to  Filoviruses – Protein Function Possible functions of sGP: How would sGP subvert immune system? 27  Filoviruses – Protein Function Minor nucleocapsid protein VP30 activates viral mRNA synthesis in Ebola virus: A stem-loop structure at the beginning of the NP gene inhibits RNA polymerase from initiating mRNA synthesis  VP30 reverses this inhibition If stem-loop is experimentally disrupted by mutations, transcription is not dependent on VP30 VP30 is present in nucleocapsids but mechanisms of action is unclear Stem-loop Fig. 16.3 structure can be present in the genome or NP mRNA Fig. 16.7 28  Filoviruses – Protein Function VP40 is the most abundant viral protein and is associated with viral envelope: Located at cytoplasmic side of plasma membrane and/or inner side of viral envelope Like matrix proteins of other enveloped viruses it bridges envelope gp’s to nucleocapsids Expression of VP40 in mammalian cells results in virus-like particles that bud from the plasma membrane Cellular proteins that are involved with trafficking and sorting of intracellular vesicles may interact with VP40 to form virions at plasma membrane e.g. Nedd4 (ubiquitin ligase), Tsg101 29 (part of multiprotein complex that directs vesicles  Filoviruses – Replication Cycle Filovirus replication cycle: Can you indicate / identify all the steps? Does this figure show Ebolavirus or Marburgvirus? How can you tell? 30  Filoviruses – Latent Infection In those who survive Ebola virus infection: Observed periodic spikes in EBOV antibodies occurring months after clinical resolution of symptoms  suggests latent infection and potential disease recrudescence that could trigger outbreaks Recrudescence: recurrence of disease / symptoms after a period of inactivity Authors suggest vaccinating survivors of Ebola virus may help prevent disease recrudescence  to boost protective antibody responses in survivors. 31  Filoviruses – Latent Infection Other recent reports showing persistence of EBOV in survivors, despite clearance from blood and clinical recovery: Semen persistence and subsequent sexual transmission Recrudescent disease resulting in severe organ-specific inflammatory syndromes in immune-privileged sites: eyes (uveitis) and brain (meningoencephalitis) Using non-human primate models, the study showed persistent EBOV infection in Lethal relapse in an Ebola virus brain of 7/36 animals that survived EBOV 32 disease (EVD) survivor that resulted infection after antibody treatment.  Filoviruses – Latent Infection Links between 2014 and 2021 outbreaks?  viral sequences recognized from 5 years after acute infection (Nature 2021) 33  Influenza Viruses - Structure Family Orthomyxoviridae from Greek ortho (correct / normal), myxa (mucus)  based on ability of virus to attach to mucoproteins on cell surface. Myxoviruses split into paramyxo and orthomyxo, with different structure and replication cycles Enveloped particles, quasi-spherical or filamentous  envelope is derived from host membrane by budding. Diameter 80-120 nm Compact helical nucleocapsids 34 EM image of Influenza Virus Model of Influenza Virus Schematic of Influenza Virus  Influenza Viruses - Types Multiple subtypes within each virus type distinguished by variations in the surface glycoproteins – hemagglutinin (HA) and neuraminidase (NA) Highly pathogenic and low pathogenic avian influenza Orthomyxoviridae have two additional genera: Thogotovirus (transmitted by ticks) Isavirus (infects fish, particularly salmon) 35  Influenza Viruses – Disease and Spread Host range of Influenza A viruses: 36  Influenza Viruses – Disease and Spread Symptoms: High fever, sore throat, cough, headache, muscular pain Most fatal in elderly, infants and chronically ill, often by secondary bacterial infections Influenza A viruses cause serious acute disease in humans, sporadic pandemics, and annual epidemics every winter  best studied 20,000 deaths/year in North America Emerging avian influenza virus strains continually threaten birds and have a potential for another human pandemic. Why weren’t there as many flu cases during COVID-19 pandemic? 37  Influenza Viruses – Disease and Spread Symptoms: High fever, sore throat, cough, headache, muscular pain 38  Influenza Viruses – Disease and Spread Influenza virus infections of the respiratory tract can lead to secondary bacterial infections (e.g. pneumonia) by providing easier access: Virus infects and causes a loss of the ciliated epithelium and disrupts mucociliary flow – flow of mucus and debris along the epithelial lining of the respiratory tract  moved by cilia on epithelial cells Virus induces production of interferons and cytokines  local and systemic inflammatory response TEM SEM 39  Influenza Viruses - Genome Segmented negative sense ssRNA genome (6-8 different segments), multiple helical nucleocapsids  single virion includes a ‘complete set’ of genome fragments. Genome size (total) ~10-15kb 40 Influenza Viruses – Genome and Proteins 41  Coming up…. Negative strand RNA viruses Influenza viruses Viruses that use reverse transcriptase HIV Midterm Exam: Oct 25th! Covers lectures 1-5. 42