Chapter 11 Lecture Outline Viral Molecular Biology

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Outline 11.1 Phage T4: The classic Molecular Model 11.4 A segmented (-) Strand RNA Virus: Influenza 11.5 A Retrovirus: Human Immunodeficiency Virus Note: We will not cover 11.2, 11.3, 11.6! Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Prokaryotic Viruses Must bind to host cell receptor Must cross a cell wall In gram hosts 2 membranes to cross Must not damage host cell initially Use host nucleotides, amino acids, ATP Replicate viral genome, build capsid, assemble new viruses Exit through cell wall Usually lyse host cells Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Bacteriophage T4 Complicated structure 170 genes 10 different capsid protein types Tail fibers bind host cell Receptor = OmpC porin Outer membrane protein Long tail injects DNA Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Phage T4 Adsorption and DNA Injection Tail fibers bind to outer membrane (ompC, LPS). Baseplate binds outer membrane Tail sheath contracts, internal tube penetrates outer membrane Injector digests peptidoglycan; internal tube penetrates cell wall Injector contacts inner membrane; DNA expelled into cytoplasm. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Phage T4 Genome Replication Upon entry, genome forms a circle Early genes transcribed Take control of cell, destroy cell chromosome Replicates via Complicated structure Uses cell nucleotides to replicate genome “Rolling circle replication” Continuous replication of many copies of genome Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Rolling Circle Concatemer Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Phage Particles Self-Assemble Late genes transcribed Capsid particles Head polymerizes around progeny DNA Tail fibers, long tail made Head, tail, tail fibers assemble Lysis protein made Destroys cell wall Releases progeny Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Summary Phage T4 Replication The phage particle attaches to the outer membrane of E. coli and injects its genome. Early genes are transcribed and translated into proteins, including nucleases to cleave host DNA and proteins for phage DNA replication. Phage DNA undergoes rolling-circle replication, generating a multigenome concatemer. Late genes are expressed to make head and tail components. Phage genomes are packaged into heads. Heads are assembled onto tails. Tail fibers are added. A phage-encoded lysozyme lyses the host cell, releasing about 200 completed phage particles. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Animal Viruses Simpler entry into cell Don’t have to cross a cell wall Can enter through endocytosis Or fusion of viral envelope to plasma membrane More complex cycle in the cells Must travel between organelles Host transcription machinery in nucleus Translation in cytoplasm Transport via ER, Golgi, endosomes Exit via cell lysis or budding Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Viral mRNA Prokaryotes: multiple proteins from 1 mRNA Operon Viral genome can be RNA or DNA Multiple proteins created Eukaryotes: 1 mRNA  1 protein Viral DNA genome gives multiple mRNAs RNA virus can create only 1 protein Solutions: 1 protein cuts itself into smaller proteins (polio) Multiple RNAs in a single virion (influenza) RNA used to make DNA  multiple proteins (HIV) Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

(-) Strand RNA Virus: Influenza Pandemic of 1918 Greatest one-year loss of life in recorded history RNA inside shell of matrix proteins Inside lipid envelope 2 major envelope proteins Neuraminidase Hemagglutinin Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

(-) Strand RNA Virus: Influenza 8 separate RNAs in genome Each encodes one protein (-) strand RNA can’t be read by ribosome Must be transcribed to (+) RNA No replicating RNA polymerase in host All (-) RNA viruses must bring own polymerase protein into host cell Influenza binds to host sialic acids Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

(-) Strand RNA Virus: Influenza (-) strand RNA moves to nucleus (+) strand mRNA synthesized Move to cytoplasm Viral proteins made Envelope proteins placed in plasma membrane (+) strand used to make progeny (-) RNA Assembly at plasma membrane Budding to release progeny viruses Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

(-) Strand RNA Virus: Influenza Viral RNA polymerases are inaccurate Introduce many mutations Antigenic drift Rapid evolution New flu virus species every year New vaccine necessary Cell infected by 2 strains can recombine Assemble new combinations of RNAs Reassortment = antigenic shift Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Human Immunodeficiency Virus Bullet-shaped capsid Encloses 2 identical copies of RNA Plus polymerase proteins Surrounded by envelope Envelope proteins embedded Binds to receptor CD4 protein Immune system T cells Microglia cells in brain AIDS-related dementia Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Human Immunodeficiency Virus Viral envelope fuses with plasma membrane Genome released into cytoplasm Polymerase replicates DNA from RNA Reverse transcriptase RNA destroyed as DNA made Forms dsDNA Acts as transposon Moves to nucleus Inserts into host genome (+) ssRNA (+) ssDNA dsDNA Integration into host genome Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Human Immunodeficiency Virus Reverse transcriptase is inaccurate Introduces many mutations Mutant viruses form within single individual Single vaccine will not protect against all forms 5 major forms of HIV in humans Many minor forms in each individual as disease progresses From integrated DNA RNA is transcribed RNA is used for progeny RNA, envelope and capsid proteins Assembles in cytoplasm Exits via budding Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Retroviruses Replicate transposon DNA Insert repeatedly in chromosome Destroy host cell Eukaryotic cells have many retroelements Old, inactivated retrovirus copies Presence of many copies allows recombination Movement of sections of chromosome Moves pieces of genes together to make new genes Major mechanism of evolution Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Animal (-) strand RNA viruses always carry into the host cytoplasm their own: genome and polymerase protein genome and envelope proteins genome and capsid proteins Answer: A Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Why is it difficult to make a vaccine against HIV? The virus mutates quickly inside an infected individual. The envelope proteins cannot be isolated in the laboratory. Different strains of HIV recombine to make new virus types. Answer: A Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.