Poxviruses Lecture 11 PDF

Lecture 11 Poxviruses Structure and classification Disease and history Genome and proteins Assembly and release 1  Poxviruses Complex, ovoid or brick shaped particles: ~ 310nm (L) x 240nm (W) x 140...

Lecture 11 Poxviruses Structure and classification Disease and history Genome and proteins Assembly and release 1  Poxviruses Complex, ovoid or brick shaped particles: ~ 310nm (L) x 240nm (W) x 140 nm (H) Surface ridges or “tubules” No typical symmetry elements Intracellular Internal core and lateral bodies Virions exist in two infectious forms: 1. Mature virus (MV): one lipid membrane MV EV 2  Poxviruses Poxviridae from English pocks (pox), referring to blistering skin lesions Two subfamilies: Chordopoxvirinae: Infects vertebrates e.g. monkeys, cattle, humans Humans: variola (smallpox), vaccinia (vaccine strain for smallpox), molluscum contagiosum (only existing natural poxvirus infection of humans; relatively rare but more common in immunocompromised patients) Entomopoxvirinae: 3  Poxviruses The two subfamilies are divided into several genera that infect multitude of hosts See https://ictv.global/taxonomy for complete taxonomy 4  Poxviruses – Disease and History Smallpox was a debilitating and fatal disease globally Smallpox scars on Egyptian mummies provide evidence for early existence Written records suggest that virus was endemic in Egypt and India by first century AD Spread throughout Asia, Europe and North Africa by the tenth century AD and later colonization spread the virus globally in 16-18th centuries 17th century: every child was expected to be exposed Mortality rates of 1- 40%, but in endemic countries average case fatality was 25% 5  Poxviruses – Disease and History Disease progression: Respiratory infection (spread via nasopharyngeal secretions or scabs/pus) Asymptomatic phase (12 days) Sudden onset fever, rash, vomiting (4 days) Painful rash, fever resumes, blisters covering body and airway, difficulty breathing and eating (12 days) 6  Poxviruses – Disease and History Variolation: infection via a non-natural route that yields less severe disease Led to developing the concept of vaccination, which has eradicated smallpox worldwide. Variolation was named after the virus the causes smallpox (variola virus) Small amount of material from smallpox sores (pustules) transferred to uninfected individuals Scratching material on the arm (cutaneous variolation) or nasally 7  Poxviruses – Disease and History 1796: Edward Jenner developed and published a procedure of vaccination (immunization) for smallpox using the material from cowpox lesions Observed that milkmaids who had gotten cowpox did not show any symptoms of smallpox after variolation But, see the links below for a different Developed cowpox lesions on story: their hands https://www.mcgill.ca/oss/article/medic al-critical-thinking-history/white-lie-he art-vaccine-history Tested inoculation with cowpox from hand of milkmaid on an 8yr https://www.um.edu.mt/umms/mmj/PD old boy & 6 weeks later F/323.pdf 8  Poxviruses – Disease and History From: https://www.mcgill.ca/oss/article/medical-critical-thinking-history/white-lie-heart-vaccine-history By mid 1800’s: the virus used to make smallpox vaccine changed from 9 cowpox  vaccinia virus  Poxviruses – Disease and History Eradication of smallpox is considered one of the biggest achievements in the international public health. 10  Poxviruses – Disease and History Poxviruses remain a subject of intense research interest Vaccinia virus is now used for smallpox (variola) vaccination and in research laboratories For eradication and safety concerns, there are limited worldwide stocks of live variola virus: 1981: Four countries were WHO collaborating centres: USA, England, Russia and South Africa 1984: England and South Africa removed their stocks Currently only two worldwide sources: CDC in Atlanta, GA and VECTOR Institute in Koltsovo, Russia 11 Smallpox virus is highly stable and old stocks can still be viable  Poxviruses – Genome and Proteins Linear double-stranded DNA, 150-250 kb (e.g. vaccinia virus: 200 kb, shown below) Covalently closed hairpin ends: no free 3’ and 5’ ends with 10 kb inverted terminal repeats 150-250 genes  up to 100 proteins in the virus! Each gene has its own transcriptional promoter No introns in the genome  no splicing required Fig. 26.1 12  Poxviruses – Genome and Proteins Virion sections show a biconcave core flanked by lateral bodies (red)  trypsin-sensitive, containing proteins (of unknown composition and function) Internal core is surrounded by core wall structure Tubular structure within the core, contains the DNA Contains breadth of viral enzymes for viral mRNA synthesis Fig. 26.4 13  Poxviruses – Genome and Proteins Two forms differ in membrane composition, as a result of differences in morphogenesis Both forms are infectious, but use a different set of viral structural proteins (surface) to bind to cellular receptors. Intracellular 14  Poxviruses – Genome and Proteins Mature virus (MV): has an outer envelope  enters by fusion or endocytosis attaches to cellular glycosaminoglycans (broad host range)  polysaccharides formed from repeating units of different 6-C sugars, including one amino sugar  bound to host surface proteins 15  Poxviruses – Genome and Proteins Extracellular virus (EV): Mature virus wrapped in an additional lipid bilayer, with a distinct set of glycoproteins Enters cells by phagocytosis  outer membrane ruptures due to decrease in pH in endosome  releases mature virus particle  MV fuses with vesicle membrane  virion core is released into cytoplasm EV envelope is fragile  can rupture once in contact with host cell 16 surface  releases MV particle   Poxviruses – Genome and Proteins Poxviruses replicate in the cytoplasm  relatively rare for DNA viruses Virus-coded enzymes packaged in the core carry out early RNA synthesis and packaging: Vaccinia virion can be considered a ‘mini-nucleus’, an mRNA synthesis machine! Viral enzymes transcribe, cap, methylate and polyadenylate mRNA 17  Poxviruses – Genome and Proteins Enzymes encoded by viral DNA also direct DNA replication: 18  Poxviruses – Genome and Proteins Poxvirus genes are expressed in a regulated transcriptional cascade controlled by viral transcription factors that bind to specific promoter sequences (~35 nt) in viral DNA: Virus early transcription factors (VETF) are packaged into virion and act upon release into cytoplasm Viral intermediate transcription factors (VITF) trigger intermediate gene expression e.g. VLTF genes Viral late transcription factors (VLTF) activate late genes  encode VETF to be packaged with progeny virions Sequential expression ensures that enough new replicated DNA is available before structural proteins are synthesized. 19  Poxviruses – Genome and Proteins Genes required for DNA replication are early genes  expressed immediately after an infection Expressed within virus cores  early mRNAs are translated into ‘early’ viral proteins Some of the early viral proteins dissolve the virus core and release viral DNA into the cytoplasm  further Fig. 20 26.5  Poxviruses – Genome and Proteins Viral DNA replication in cytoplasm forms “DNA factories” visible under EM (viroplasm) 21  Poxviruses – Genome and Proteins Poxviruses produce large DNA concatemers (oligomers of genome length units) that are later resolved into monomers Concate mer junction 22  Poxviruses – Genome and Proteins Poxviruses produce large DNA concatemers (oligomers of genome length units) that are later resolved into monomers Cruciform extrusions = concatemer junctions Concatemers are resolved by virus- coded resolvase 23  Poxviruses – Genome and Proteins Poxviruses produce large DNA concatemers (oligomers of genome length units) that are later resolved into monomers 1. Site-specific nick in one of the hairpins 2. Extension of the 3’ end (~100 nt) 3. After extension, hairpin ends are refolded 4. Hairpins from step 3 can be used for further DNA synthesis 5. Entire genome length is synthesized and then loops back through the 2nd hairpin  generates head-to- head dimer 6. Concatemers are resolved by virus-coded resolvase  makes staggered single strand breaks at the ends Fig. 24  Poxviruses – Genome and Proteins Intermediate and late genes are called postreplicative genes  transcribed only after DNA replication has started Uses distinct promoters and initiation factors Initiation of intermediate genes requires two VITFs and a cellular factor involved in mRNA metabolism Initiation of late genes requires three VLTFs and may require an additional early gene product as well as a cellular factor Postreplicative mRNAs have a poly(A) extensions and 3’ end heterogeneity A slippage mechanism during transcription initiation adds “poly(A) heads” 25  Poxviruses – Assembly and Release Assembly of vaccinia virions is a complex process Rigid, crescent-shaped membrane structures are synthesized (de novo synthesis) and are destined to become the mature viral envelope (not connected to a pre-existing internal membrane!?) Lipids are delivered to crescents from the ER as small vesicles (micelles) containing viral envelope proteins. 26 Fig.  Poxviruses – Assembly and Release Crescents mature into spheres (i.e. immature virions, IV in fig 26.5) and enclose viroplasm  electron dense and containing viral core proteins Viral DNA enters the spheres to form discrete and dense cores (aka nucleoids) Lipids are delivered to crescents from the ER as small vesicles (micelles) containing viral envelope proteins. 27 Fig.  Poxviruses – Assembly and Release Immature virions develop internal core and lateral bodies by rearrangement of materials captured inside the spheres  acquires final shape  mature virion (MV) Vast majority of new virions remain as intracellular MVs A small proportion of MVs undergo further maturation to become extracellular virus (EV), designed for export and cell-to-cell spread Lipids are delivered to crescents from the ER as small vesicles (micelles) containing viral envelope proteins. 28 Fig.  Poxviruses – Assembly and Release MVs can be wrapped in Golgi-derived cisternae  adds additional lipid layers and viral proteins Wrapped viruses (WV) have two bilayer membranes and are transported to the plasma membrane by actin tails The outermost viral membranes fuse with cell membrane, releasing extracellular virus (EV)  EV can remain attached to cell membrane via microvilli projections Vaccinia virus (green) moves in the 29 cell through modifying host cell actin Fig.  Poxviruses – Assembly and Release Vaccinia viruses (and some others) produce actin ‘comet’ tails  induced by viral proteins that promote actin polymerization / depolymerization  used for ‘propulsion’ / budding out of the host cell. See: 30 https://www.youtube.com/watch?v=lOblMwjwG-s  Poxviruses – Assembly and Release Poxviruses make several proteins that target host defences against invading pathogens: 31  Poxvirus – Life Cycle Can you explain the life cycle? 32  Coming up…. Vaccines and Antivirals - Last lecture! Vaccines (Chapter 35) Antivirals (Chapter 36) Quiz 5 available Nov. 27th at 10 PM until Nov. 29th, 10 PM Covers lectures 9 and 10. 33