Course in Virology Rotterdam 2018 VIRUSES IN DIFFERENT SHAPES AND FLAVORS REPLICATION STRATEGIES THE VIRAL LIFE CYCLE Raoul J. de Groot Division of Virology Faculty of Veterinary Medicine Utrecht University VIRUS CLASSIFICATION BALTIMORE CLASSIFICATION central phenomenon – de novo mRNA synthesis • HOST PLANT / ANIMAL / BACTERIUM • MORPHOLOGY NAKED OR ENVELOPED SPHERICAL OR FILAMENTOUS VII NUCLEOCAPID: HELICAL OR ICOSAHEDRAL • GENOME DNA OR RNA SINGLE OR DOUBLE STRANDED LINEAR OR CIRCULAR SEGMENTED OR NON-SEGMENTED • REPLICATION ‘BALTIMORE’ CLASSIFICATION STRATEGY REPLICATION IN EUKARYOTIC CELLS A DIVERSITY OF VIRUSES a variety of replication sites ONE GENERAL REPLICATION SCHEME • ALL VIRUSES PACKAGE THEIR GENOME INSIDE A PARTICLE THAT MEDIATES TRANSMISSION FROM HOST TO HOST • THE VIRAL GENOME CONTAINS THE INFORMATION FOR INITIATING AND COMPLETING AN INFECTIOUS CYCLE WITHIN A HOST CELL 1 THE VIRAL LIFE CYCLE ENTRY the six steps… • Attachment • Penetration • Uncoating FIRST CONTACT: ADSORPTION ENTRY REQUIRES BREACHING OF THE PLASMA MEMBRANE IN ORDER TO INTRODUCE THE VIRAL GENOME IN THE CYTOPLASM • virions equiped with metastable proteins • enveloped viruses: fusion proteins SARS coronavirus: ACE2 MERS coronavirus: DPP4 • naked viruses: pore-forming proteins Influenza virus: sialic acid • membrane fusion/pore formation requires conformational changes in virion (proteins) • triggered by the appropriate cue CELL CORTEX HOW TO OVERCOME THE CELL CORTEX? • plasma membrane-associated cytoskeleton • gel-like mesh of actin filaments, at the perimeter of the cell • mechanical strength – cell surface movement • a barrier to virus entry Sleeping Beauty – Hedge of Thorns Julius Diez (~1900) 2 A VARIETY OF ENDOCYTIC PATHWAYS ENTRY VIA ENDOCYTOSIS: SEMLIKI FOREST VIRUS TO ENTER CELLS Lopez, S. & Arias, C. (2010) How Viruses Hijack Endocytic Machinery. Nature Education Parton, R. G. & Simons, K. (2007) The multiple faces of caveolae. Nature Reviews Molecular Cell Biology ENVELOPED VIRUSES: ROUTES OF ENTRY HERPESVIRUSES – BI-DIRECTIONAL TRAFFICKING OF CAPSIDS ALONG UNIPOLAR AXONAL MICROTUBULES Scale drawing of an herpesvirus capsid, transported along a microtubule by dynein motors ENTRY The Viral Life Cycle ATTACHMENT – PENETRATION - UNCOATING • transport along microtubular network • delivery of virions in endosomes and subviral particles deep in the interior of the cell, in the perinuclear region • optimal site for virus replication • triggering of fusion proteins to induce escape from endosome (cues: a.o. low pH, proteolytic cleaveage) 3 VIRAL REPLICATION….. BALTIMORE CLASSIFICATION True DNA viruses: replication via DNA intermediates Parvoviridae Retroviridae Hepadnaviridae • Transcription VII • Translation Herpesviridae • Genome replication Reoviridae Coronaviridae Picornaviridae Togaviridae Orthomyxoviridae Paramyxoviridae Rhabdoviridae VIRAL REPLICATION….. BALTIMORE CLASSIFICATION true RNA viruses: replication via RNA intermediates Parvoviridae • Most DNA viruses replicate in the nucleus Retroviridae Hepadnaviridae VII exception: poxviruses (cytoplasm) Herpesviridae • Most DNA viruses possess a ds genome exception: parvovirus (linear single strand) Reoviridae Coronaviridae Picornaviridae Togaviridae Orthomyxoviridae Paramyxoviridae Rhabdoviridae RNA VIRUSES: GENOME POLARITY • Positive strand viruses: genome is mRNA sense: can be translated • Negative strand viruses: genome is anti-sense: cannot be translated genome non-infectious – RdRp in virion • Ambisense genome: contains genes in both orientations 4 BALTIMORE CLASSIFICATION retroviruses: replication via DNA intermediates Parvoviridae Retroviridae Hepadnaviridae VII Herpesviridae Reoviridae Coronaviridae Picornaviridae Togaviridae Orthomyxoviridae Paramyxoviridae Rhabdoviridae Retroviridae Subfamily Characteristic members Oncovirinae Oncogenic viruses Genera Oncornavirus B Leukemia virus Rous Sarcoma virus Oncornavirus C Mouse mammary Tumor virus Mason-Pfizer Monkey virus Oncornavirus D Lentivirinae Human Immuno deficiency virus Feline Immuno deficiency virus Visna virus The HIV-1 capsid pore is strongly electropositive and recruits dNTPs with rapid association and dissociation kinetics D A Jacques et al. Nature 1–5 (2016) doi:10.1038/nature19098 5 VIRAL GENES AND GENE EXPRESSION Two types of proteins: Structural and non-structural ARE REPLICATION ENZYMES NON-STRUCTURAL PER SE? structural proteins end up in the virion non-structural proteins: replication enzymes accessory proteins VIRAL GENES AND GENE EXPRESSION VIRAL GENES AND GENE EXPRESSION Two types of proteins: Essential and non-essential Viral genes are expressed to produce either: • Essential proteins: absolutely required • Individual proteins (1 mRNA, 1 protein) for replication in tissue culture cells replication enzymes • Polyproteins virion proteins processing by viral and/or host proteinases • Non-essential proteins: dispensible in vitro accessory proteins SYNTHESIS AND PROTEOLYTIC PROCESSING OF THE POLIOVIRUS POLYPROTEIN VIRAL REPLICATION TYPICALLY OCCURS IN MEMBRANOUS VIRUS-INDUCED COMPARTMENTS “VIRAL FACTORIES” 6 3D-ANALYSIS OF FLOCK-HOUSE VIRUS RNA CORONAVIRUS RNA SYNTHESIS ASSOCIATED REPLICATION COMPLEX REVEALS WITH ER-DERIVED DOUBLE MEMBRANE VESICLES A VIRUS-INDUCED MINI-ORGANELLE Kopek et al. (2007) PLOS Biol. Knoops et al. (2008) PLOS Biol. TWO TYPES OF VIRAL FACTORIES? VIRAL FACTORIES: ALSO SITES OF VIRION ASSEMBLY Flock-house virus Poliovirus Rubella virus SARS coronavirus Potato virus X virion “cages” encasing the X-body. Dengue virus Hepatitis C virus Linnik et al. (2013) Front. Plant Sci. Paul and Bartenschlager (2013) Unraveling the structure of viral replication complexes at super-resolution World J. Virol. The Viral Life Cycle VIRAL ASSEMBLY.... • production of virions • naked viruses: assembly in cytoplasms release through cell lysis 7 VIRAL ASSEMBLY.... • production of virions • enveloped viruses: budding release from intact cells BUDDING • stage of virus assembly during which the membrane is acquired • wrapping of NC in a cellular membrane, modified by virus-specific envelope proteins 8 Budding Retroviruses Fission - Maturation Viruses may differ in the location of the budding site REPLICATION STRATEGIES THE VIRAL LIFE CYCLE • insight into the diversity of viruses and their replication strategies • classification of viruses • six steps of the viral life cycle • general impression of the processes that occur during each of these steps 9.