Viruses 1 Principles of Virology, ASM Press 2 We live in a cloud of viruses • Viruses infect all living things • We eat and breathe billions of virions regularly • We carry viral genomes as a part of our own genetic material 3 How ‘infected’ are we? • HSV-1, HSV-2, VZV, HCMV, EBV, HHV-6, HHV-7, HHV-8 • Once infected, always infected 4 A retrovirus makes chicken eggshells blue Virome 5 The human virome in nonpathogenic conditions Most identified DNA sequences floating in our blood plasma belong to viruses. Amazingly, the vast majority of the viruses that infect us have little or no impact on our health 6 Not all viruses make you ill... 7 Good viruses 8 An enterovirus can replace the beneficial function of commensal bacteria Murine norovirus (MNV) infection of germ-free or antibiotic-treated mice restored intestinal morphology and lymphocyte function without inducing overt inflammation and disease. 9 10 The virus and the virion A virus is an organism with two phases 11 Infected cell virion Viruses replicate by assembly of preformed components into many particles. They make the parts and assemble the final product. Not binary fission like cells. 12 Immunization • Variolation - China (11th century), Lady Montagu (1700s) • No knowledge of agent • Survivors of smallpox protected against disease • 1790s - experiments by Edward Jenner in England establish vaccination 13 Concept of microorganisms • Leeuwenhoek (1632 - 1723) • Pasteur (1822 - 1895) • Koch (1843-1910) 14 Virus discovery • Virus discovery - filterable agents • 1892 - Ivanovsky • 1898 - Beijerinck: contagium vivum fluidum • Virus: slimy liquid, poison • 1898 - Loeffler & Frosch - agent of foot & mouth • Key concept: agents not only small, but replicate only in the host, not in broth • disease is filterable by 0.2 micron filters (µm, one millionth of a meter) 15 There is an underlying simplicity and order to viruses because of two simple facts • All viral genomes are obligate molecular parasites that can only function after they replicate in a cell • All viruses must make mRNA that can be translated by host ribosomes: they are all parasites of the host protein synthesis machinery 16 Some important definitions • A susceptible cell has a functional receptor for a given virus - the cell may or may not be able to support viral replication • A resistant cell has no receptor - it may or may not be competent to support viral replication • A permissive cell has the capacity to replicate virus - it may or may not be susceptible • A susceptible AND permissive cell is the only cell that can take up a virus particle and replicate it 17 • Animal viruses at first could not be routinely propagated in cultured cells • Most viruses were grown in laboratory animals 18 Studying the infectious cycle in cells • Not possible before 1949 (animal viruses) • Enders, Weller, Robbins propagate poliovirus in human cell culture primary cultures of embryonic tissues • Nobel prize, 1954 19 20 21 • HeLa cells (Human cervical cells infected by HPV18) – the first `immortal` cell line known to researchers, isolated in 1951 • Scientists have grown about 20 tons of her cells (2010) Batts DW (2010-05-10) • Used for the study of: • Polio – enabled vaccine development • Cancer, HIV….. • More than 11000 patents… 22 Hayflick limit Virology breakthrough in the 1950’s: The viral nucleic acid genome is the genetic code • Hershey-Chase experiment with phage T4 23 24Alfred Hershey & Martha Chase, 1952 25 Information NOT contained in viral genomes: - No genes encoding the complete protein synthesis machinery (AARS, eIFs, tRNAs) - No genes encoding proteins involved in energy production or membrane biosynthesis - No classical centromeres or telomeres found in standard host chromosomes 26 What information is encoded in a viral genome? Gene products and regulatory signals for: - Replication of the viral genome - Assembly and packaging of the genome - Regulation and timing of the replication cycle - Modulation of host defenses - Spread to other cells and hosts Smallest known viral genomes 27 Largest known viral genomes • The biggest surprise: thousands of different virions, seemingly infinite complexity of infections • But a small number of viral genome types • Viral genomes must make mRNA that can be read by host ribosomes • All viruses on the planet follow this rule, no exception to date 28 29 David Baltimore (Nobel laureate) used this insight to describe a simple way to think about viral genomes Knowing only the nature of the viral genome, one can deduce the basic steps that must take place to produce mRNA I - dsDNA II - ssDNA III - dsRNA IV - ss (+) RNA V - ss (-) RNA VI - ss (+) RNA with DNA intermediate VII - gapped dsDNA The seven classes of viral genomes Definitions • mRNA (ribosome ready) is always the plus (+) strand • DNA of equivalent polarity is also the (+) strand • RNA and DNA complements of (+) strands are negative (-) strands • Not all (+) RNA is mRNA! 30 31 DNA genomes • Transcription is the first biosynthetic reaction to occur in cells infected with dsDNA viruses • Viral DNA replication always requires synthesis of at least one viral protein, sometimes many - hence it is always delayed after infection 32 I. dsDNA genomes 33 Genomes copied by host DNA polymerase Genomes encode DNA polymerase II. ssDNA genomes 34 TT virus (ubiquitous human virus) B19 parvovirus (fifth disease) RNA genomes • Cells have no RNA-dependent RNA polymerase (RdRp) • RNA virus genomes encode RdRp • RdRp produce RNA genomes and mRNA from RNA templates 35 RNA directed RNA synthesis RNA in the virus particle • (-) strand RNA genomes: coated with protein • (+) strand RNA genomes: naked (exceptions: retrovirus, coronavirus) • dsRNA genomes 36 Universal rules for RNA-directed RNA synthesis • RNA synthesis initiates and terminates at specific sites on the template • RdRp may initiate synthesis de novo (like cellular DdRp) or require a primer • Other viral and cell proteins may be required • RNA is synthesized by template-directed stepwise incorporation of NTPs, elongated in 5’- 3’ direction • Non-templated RNA synthesis 37 38 III. dsRNA genome 39 Rotavirus (human gastroenteritis) There is no incorporation into host genome IV. ssRNA: (+) sense 40 V. - ssRNA, (-) sense 41 VI. ssRNA(+) sense with DNA intermediate 42 http://hyperphysics.phy- astr.gsu.edu/hbase/organic/dogma.html One viral family: Retroviridae Two human pathogens: Human immunodeficiency virus (HIV) Human T-lymphotropic virus (HTLV) Reverse transcriptase 43 • Primer can be DNA or RNA • Template can be RNA or DNA • Only dNTPs, not rNTPs, are incorporated RT • Bacteria and Archaea have RT activity • Therefore RT evolved before the separation of Archaea, bacteria, and eukaryotes • RT might be the bridge between early RNA world and modern DNA world • RT also in HBV, Caulimoviridae • One DNA produced from two RNAs by RT • Strong promoter (the LTR) built during RT • Proviral DNA directs the host transcription machinery to synthesize many copies of viral mRNA • Viral mRNA is translated into viral proteins OR encapsidated into virus particles There is no DNA replication and no RNA replication 44 DNA synthesis: cytoplasmic VII. Gapped dsDNA genomes 45 Hepadnaviridae Hepatitis B virus Reassortment: Consequence of segmented genome 46 Ambisense RNA genomes 47 Universal rules of DNA replication • DNA is synthesized by template-directed incorporation of dNMPs into 3’-OH of DNA chain • DNA is always synthesized 5’-3’ via semiconservative replication (two daughter strands) • Replication initiates at specific sites on template called origins • Catalyzed by DdDp + accessory proteins • Always primer-dependent 48 What’s the host for? Viruses can’t do it themselves • Viral DNA replication always requires synthesis of at least one viral protein, sometimes many (hence always delayed after infection) • Simple viruses require more host proteins genetic economy • Complex viruses encode many, but not all proteins required for replication 49 Viral proteins • DNA polymerase and accessory proteins • Origin binding protein, helicases • Exonucleases • Enzymes of nucleic acid metabolism (thymidine kinase, ribonucleotide reductase, dUTPase) 50 Strategies of transcription of viral DNA 51 There are three types of genes: immediate-early, early and late. The immediateearly genes are transcribed immediately after infection and ensure the transcription of early genes, which encode the proteins necessary for the viral replication. The late genes mostly encode structural proteins. Standing apart are genes expressed during latency. Engineering mutations into viral genomes - the modern way 52  Infectious DNA clone: transfection  A modern validation of the Hershey-Chase experiment (1952)  Deletion, insertion, substitution, nonsense, missense Transfection  - Production of infectious virus after transformation of cells by viral DNA, first done with bacteriophage lambda  - Transformation-infection We live and prosper in a cloud of viruses 53 • Most infections have no consequence • If we do get infected, many infections are inapparent Viral pathogenesis • Pathogenesis: the process of producing a disease • Two components of viral disease: - Effects of viral replication on the host - Effects of host response on virus and host Three requirements for a successful infection • Sufficient amount of virus • Cells accessible, susceptible, permissive • Local antiviral defense absent or overcome Gaining access: site of entry is critical • The human body presents only a limited spectrum of entry sites for viral infection. 54 Transmission of infection • Spread of infection from one susceptible host to another; required to maintain chain of infection Transmission terms • Horizontal transmission - between members of same species (zoonotic different species) • Iatrogenic - activity of health care worker leads to infection of patient • Nosocomial - when an individual is infected while in hospital or health care facility • Vertical transmission - transfer of infection between parent and offspring • Germ line transmission - agent is transmitted as part of the genome (e.g. proviral DNA) 55 Virulence • Capacity of a virus to cause disease in a host • Virulent vs avirulent or attenuated virus • Virulence can be quantitated: - Mean time to death - Mean time to appearance of symptoms - Measurement of fever, weight loss - Measurement of pathological lesions (poliovirus); reduction in blood CD4+ lymphocytes (HIV-1) Viral virulence is a relative property • Influenced by dose, route of infection, species, • age, gender, and susceptibility of host • Cannot compare virulence of different viruses • For similar viruses, assays must be the same 56 Virulence depends on route of inoculation Lymphocytic choriomeningitis virus Acute vs persistent infections • Acute infection - rapid and self-limiting • Persistent infection - long term, for the life of host • Stable, characteristic for each virus family • Most persistent infections probably begin as an acute infection 57 Virus particles are metastable • Must protect the genome (stable) • Must come apart after infection (unstable) 58 Functions of structural proteins Protection of the genome - Assembly of a stable protective protein shell - Specific recognition and packaging of the nucleic acid genome - Interaction with host cell membranes to form the envelope Delivery of the genome - Bind host cell receptors - Uncoating of the genome - Fusion with cell membranes - Transport of genome to the appropriate site How is metastability achieved? • Stable structure - Created by symmetrical arrangement of many identical proteins to provide maximal contact • Unstable structure - Structure is not usually permanently bonded together - Can be taken apart or loosened after infection to release or expose genome 59 Viruses are obligate intracellular parasites Virus particles are too large to diffuse across the plasma membrane 60 Finding the ‘right’ cell • Step 1: adhere to cell surface (electrostatics) - No specificity • Step 2: Attach to specific receptor molecules on cell surface - More than one receptor may be involved • Step 3: Transfer genome inside the cell Cellular receptors for viruses • Essential for all viruses except those of fungi (no extracellular phases) and plants (enter cells by mechanical damage) • 1985: one receptor known, sialic acid for influenza virus • Different viruses can bind the same receptor • Viruses of the same family may bind different receptors 61 Entry into cells 62 63 Assembly is dependent on host cell machinery • Cellular chaperones • Transport systems • Secretory pathway • Nuclear import and export machinery Viral proteins have ‘addresses’ • Membrane targeting: Signal sequences, fatty acid modifications • Membrane retention signals • Nuclear localization sequences (NLS) • Nuclear export signals 64 Sub-assemblies • Ensure orderly formation of viral particles and virion subunits • Formation of discrete intermediate structures • Can’t proceed unless previous structure is formed: quality control 65 Three strategies for making sub- assemblies A – Assembly from individual protein molecules 66 67 Genome packaging • Problem: Viral genomes must be distinguished from cellular DNA or RNA molecules where assembly takes place • Solution: Packaging signals in the viral genome 68 Also capsid limits the amount of genetic material Darwin would have loved viruses! 69 The best exemplars of evolution by natural selection, and for RNA viruses, evolution is so rapid it can be followed in real time Modern virology has provided a window on the mechanisms of evolution • As host populations grow and adapt, virus populations are selected that can infect them - New viral populations emerge every day It also works the other way - Viral populations can be significant selective forces in the evolution of host populations • If a host population cannot adapt to a lethal virus infection, the population may be exterminated 70 The public is constantly confronted with the reality of viral evolution (even if they don’t believe in evolution) • New viral diseases: AIDS, West Nile virus in the US, HCV, Ebolavirus, Zika virus • Regular bouts every year with influenza and common cold viruses • Drug resistant HIV • Simple fact: viruses evolve faster than many can comprehend 71 The interface of host defense and virus replication is fertile ground for selection and evolution 72 Replicating viruses produce large numbers of mutant genomes • Evolution is possible only when mutations occur in a population • Mutations are produced during copying of any nucleic acid molecule Viral genomes are always mutating! RNA viruses Lack of proofreading activity in RNA dependent RNA polymerase: high error frequencies (1 misincorporation / 10 3 - 10 4 nt polymerized) • Average error frequency: 1 in 10 4 or 10 5 nucleotides polymerized • In a 10 kb RNA virus genome, a mutation frequency of 1 in 10 4 results in about 1 mutation per genome DNA viruses • Genome replication not as error prone as RNA viruses • Proofreading • Most DNA viruses generate less diversity, evolve slower than RNA viruses The quasispecies concept For a given RNA virus population, the genome sequences cluster around a consensus or average sequence, but virtually every genome can be different from every other It is unlikely that a genome with the consensus sequence is actually replicating in the population 73 The myth of consensus genome sequences • Analysis of an RNA bacteriophage population (Qβ): “A Qβ phage population is in a dynamic equilibrium with viral mutants arising at a high rate on the one hand, and being strongly selected against on the other. The genome of Qβ cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.” E. Domingo, D. Sabo, T. Taniguchi, C. Weissmann. 1978. Nucleotide sequence heterogenity of an RNA phage population. Cell 13:735-744. 74 Quasispecies • Variation further generated by recombination and reassortment 75 • Survival of the fittest: A rare genome with a particular mutation may survive a selection event, and this mutation will be found in all progeny genomes • Survival of the survivors: However, the linked, but unselected mutations, get a free ride • Consequently, the product of selection after replication is a new, diverse population that shares only the selected mutations Selection Diversity is selected Mutations in viral polymerases that reduce the frequency of incorporation errors: - Do not have a selective advantage when wild type and anti-mutators are propagated together - Lower rates are neither advantageous nor selected in nature - Mutants are often less pathogenic 76 • High mutation rates are selected during virus evolution: mutation is good for viral populations Fitness decline compared to initial virus clone after passage through a bottleneck 77 78 Virus-host conflicts have driven evolution of the immune system 79 Mechanisms of drug resistance • RNA viruses: error prone RNA polymerase, no correction mechanism • One misincorporation in 104 - 105 nucleotides polymerized (106 greater than host DNA genome) • In RNA viral genome of 10 kb, this frequency leads to one mutation in 1-10 genomes • DNA viruses: most DNA polymerases can excise and replace misincorporated nucleotides • DNA viruses evolve more slowly than RNA viruses because they have less diversity 80 Despite this genome diversity… • There are only 3 serotypes of poliovirus, but >150 of rhinoviruses • One measles serotype, continuous influenza variation • Why? 81 Selection: Is virulence a positive or negative trait? • Idea: increased virulence reduces transmissibility because hosts die faster, reducing exposure to uninfected hosts • Expectation: all viruses evolve to be maximally infectious and avirulent • But this is not observed - there are many virulent viruses 82 The fundamental properties of viruses constrain and drive evolution • Despite many rounds of replication, mutation, selection, we can recognize a herpesvirus or influenza virus genome by sequence analysis • Viral populations often maintain master or consensus sequences, despite opportunities for extreme variation • How is stability maintained? Constraining viral evolution: • Extreme alterations in viral consensus genome do not survive selection, the viral genome is one constraint - DNA cannot become RNA, or vice versa - Replication strategy - cannot change; consider interaction with host proteins • Physical nature of capsid - Icosahedral capsids: defined internal space, fixes genome size • Selection during host infection - A mutant too efficient in bypassing host defenses will kill host, suffer the same fate as one that does not replicate efficiently enough 83