Microbiology- a clinical approach by Anthony Strelkauskas et al Chapter 12: The structure and infection cycle of viruses

 More than 80% of infectious diseases are caused by viruses.  As a health professional, most infectious disease that you see will be caused by viruses.  It is important that you have an understanding of viral structure and the infection cycle cause by viruses.

 Emerging infectious diseases ◦ Infectious diseases whose incidences have increased over the past 30 years or threaten to increase ◦ Includes new pathogens not observed before  Mutated viruses ◦ Includes diseases whose etiology was not established before  Re-emerging infectious diseases ◦ Infectious diseases we once thought to have overcome bounce back

 Viruses are acellular.  They are obligate intracellular parasites.  Viral genome subverts host cell’s machinery to reproduce.  Viruses depend on host metabolism. ◦ Energy, materials, enzymes  Viruses infect living cells. ◦ Bacteria (called bacteriophages) and archaea ◦ Plant cells ◦ Animal cells (human cells included in this group).  They are specific for a certain cell type.

 They are composed of nucleic acids and proteins. ◦ Their genome is either RNA or DNA ◦ Their genome can be single stranded (ss) or double stranded (ds)  Viral nucleic acid is surrounded by a protein coat called a capsid. ◦ Each capsid is made up of capsomeres.  Based on the shape of the capsid viruses are classified as  Some viruses are enclosed by a membrane donated from the host ◦ Enveloped viruses ◦ Naked viruses do not have an envelope.  An intact, complete viral particle is called a virion. Icosahedral (spherical)Helical (Filamentous) Complex

donated from host

 Their shape is derived from 20 triangular faces that make up the symmetric capsid.  Capsid contains spikes, glycoproteins used to attach to the host cell. naked virus, no envelope

 Contains 8 segments of RNA enclosed in nucleoprotein  Has an additional layer of protein = matrix protein  Has an envelope with hemagglutinin (for binding to host cell in initial infection) and neuraminidase (for release of newly made virions) embedded

 Mixture of icosahedral and helical shapes ◦ Many bacteriophages  Asymmetrical (irregular) shapes ◦ Tend to be larger viruses  Poxviruses

 Many viruses that infect humans and other animals are enveloped.  Envelopes originate from the host plasma membrane or membranes of host organelles into which viral proteins/glycoproteins have been embedded. ◦ All envelopes have a phospholipid bilayer. ◦ Viral proteins/glycoproteins often serve attachment to new host cells and are involved in the infection cycle.

 Genome packaging has an important role in the infection.  Viral genomes are packaged in various ways including: ◦ Directly in the capsid ◦ Associated with special nucleic acid binding proteins (often accompanied by additional proteins that connect the nucleocapsid to the plasma membrane if enveloped)

Other classification schemes: By host cell By the way how the virus genome is replicated (Baltimore classification)

 The infection cycle was first worked out in bacteriophages (bacterial viruses). ◦ Lytic : leading to bacterial cell death ◦ Lysogenic : virus genome is inserted into bacterial genome; can be reactivated and funneled into the lytic cycle  Animal virus infections can be also either lytic leading to host cell death or become latent by incorporation into the host genome.

 In a lytic infection, the host cells fills with virions and bursts. ◦ The result is cell death.  Lysogenic infections are also known as latent infections. ◦ The viral genome (in DNA) becomes incorporated into the host cell ’ s DNA (provirus).  It can remain this way for an extended period.  The host cell lives.  Upon cell division the viral genome is also duplicated. ◦ Reactivation and active virion production is possible depending on external stimuli.

 Movie clips\12.01_Lytic_Lysogenic_Cycles.mov Movie clips\12.01_Lytic_Lysogenic_Cycles.mov

 For animal viruses, there are six steps in lytic infection: ◦ Attachment ◦ Penetration/uptake ◦ Uncoating ◦ Biosynthesis ◦ Maturation ◦ Release → Specific for animal virus, absent in bacteriophages

 Attachment occurs when a virion binds to specific receptors on a host cell.  Some viruses require a co-receptor  Without the co-receptor, there is no infection.

 Many different host cell molecules can be used as viral receptors. ◦ Some viruses use more than one type. ◦ Some receptors are shared by many viruses. ◦ Receptors can determine host range of virus.  Virus-receptor binding is high affinity. ◦ There are conformational interactions.  Non-enveloped viruses ◦ Binding takes place between viral capsid (spikes!) and receptor.  Enveloped viruses ◦ Binding takes place between viral envelope proteins and receptor.

 Once attached, the virus must gain entry to the host cell.  It must also uncoat or remove the capsid.  Uncoating can occur in three places: ◦ At the plasma membrane ◦ In the cytoplasm ◦ At the nuclear membrane

Non-enveloped virusesEnveloped viruses  Use receptor mediated endocytosis to gain entry into the host cell ◦ Virus is enclosed in a vesicle – the endosome ◦ Endosomes fuse with the lysosome where uncoating begins.  Some viruses form a pore in the host membrane.  Envelope fuses with the host cell membrane ◦ Fusion is mediated by specialized fusion proteins.  For some viruses, fusion requires the presence of co-receptor molecules.

 Viral infection requires compartmentalization. ◦ Viral genomes, capsids, and other viral proteins are synthesized in specific locations in the host cell. ◦ Newly synthesized viral components are moved to other locations for assembly of viral particles.  Viral components are moved in vesicles, using host cell microtubules.  DNA viruses or retroviruses transport their genome into the nucleus.

 Biosynthesis of new viral components can be complex. ◦ Viral genomes are either DNA or RNA. ◦ Both can be single or double-stranded. ◦ Single stranded RNA viruses can be of (+) or (-) RNA ◦ Retroviruses have an RNA genome which is converted into DNA during the replication process.  If possible the host cell machinery is used.  Host DNA synthesis is inhibited by the virus. ◦ All polymerases and proteins concentrate on viral DNA synthesis.

 DNA viruses must go to cell nucleus to use host polymerase ◦ Or replicate in cytoplasm with viral polymerase  RNA viruses must encode a viral polymerase ◦ Mostly packaged in the virion  dsRNA and (+)ssRNA genome can be directly translated  (-)ssRNA and retrovirus genomes must be replicated to be able to be translated

 Double-stranded DNA viruses use the same mechanisms as the host cell for biosynthesis. ◦ One strand of viral DNA is transcribed into mRNA.

 Viral strand is used as a template to make a complementary copy of DNA. ◦ This uses the host cell ’ s DNA polymerase. ◦ The complementary copy is transcribed into mRNA. ◦ It is also used to make new copies of the viral genome.

 Latent viruses do not kill host cells.  The viral genome is inserted into a host chromosome.  Maximum replication is not required. ◦ A small number of viral genes are expressed. ◦ A limited number of viral genomes are replicated.

 Mechanisms of biosynthesis are more complicated than in DNA viruses.  Host cells do not possess RNA-dependent polymerases. ◦ They are required to make viral mRNA and replicate genomes. ◦ Viruses must carry one.

 Genomes contain one (+) strand and one ( – ) strand.  During infection, the ( – ) strand is copied into messenger RNA. ◦ This is done by a viral RNA polymerase. ◦ The mRNA is used to produce viral proteins.  Each of the strands is used as a template to make a new double-stranded genome.

 The (+) strand is already mRNA. ◦ It can be directly translated into viral proteins.  Genome replication has 2 steps: ◦ The (+) strand is copied into ( – ) template ◦ The template is used to make more (+) strands.

 The ( – ) strand cannot be used as mRNA ◦ It must first be copied into a (+) template strand. ◦ This is done using a viral RNA polymerase packaged with the virus.  The new template strand is mRNA: ◦ It is used to synthesis viral proteins. ◦ It is also used to make new viral genomes

 Retroviruses are RNA viruses that contain the enzyme reverse transcriptase. ◦ Reverse transcriptase converts RNA into DNA. ◦ Converted viral DNA can be inserted into the host cell chromosome.  Retroviruses cause latent infections.

 Movie clips\12.02_Biosynthesis1_dsDNA.mov Movie clips\12.02_Biosynthesis1_dsDNA.mov  Movie clips\12.03_Biosynthesis2_ssDNA.mov Movie clips\12.03_Biosynthesis2_ssDNA.mov  Movie clips\12.04_Biosynthesis3_dsRNA.mov Movie clips\12.04_Biosynthesis3_dsRNA.mov  Movie clips\12.05_Biosynthesis4_+ssRNA.mov Movie clips\12.05_Biosynthesis4_+ssRNA.mov  Movie clips\12.05_Biosynthesis4_-ssRNA.mov Movie clips\12.05_Biosynthesis4_-ssRNA.mov

 Intracellular trafficking ◦ Involves the movement of newly made viral components to specific sites in the host cell. ◦ Transported through the cell by host cell microtubules  Assembly ◦ Site depends on  The type of genome (DNA or RNA)  The mechanism of genome replication  The presence or absence of an envelope.

 All virions must complete a common set of assembly reactions. ◦ Formation of structural subunits for the capsid ◦ Assembly of the capsid ◦ Association of viral genome within the capsid ◦ Enveloped viruses: assembly of viral envelope glycoproteins.

 New virions can be released from the host cell in two ways: ◦ Lysis (non-enveloped viruses) ◦ Budding (enveloped viruses) Budding of influenza virus

 Viruses can spread from cell to cell. ◦ They can use tight junctions between cells (b, c). apical basolateral

 Viruses can spread from cell to cell. ◦ They can also spread through the formation of syncytia. ◦ Syncytia are fused giant cells filled with virus. 87 h pi

 Some viruses produce decoy virions. ◦ These are empty capsids or non-infectious virions ◦ They confuse and distract the host defenses.  Some viruses incorporate host proteins as a type of camouflage.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 40  Viruses must be cultured within a host cell ◦ Bacteria in culture ◦ Animal cells in tissue culture or eggs  Host cells form confluent lawn ◦ Viruses form plaques where host cells were lyzed  Plaque is a colony of viruses Bacterial lawn Confluent animal cells

 Eclipse: not a single intact virion present  Latent period: No extracellular virions detectable Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Rise period: Release of virus progeny Burst size: total number of progeny released per infected cell.

 Viral structures include nucleic acids (either DNA or RNA), and a capsid protein coat made of capsomere subunits.  Some viruses are surrounded by envelopes composed of viral glycoproteins, oligosaccharides, and host cell membrane lipids.  Viruses may lyse the host cells by using a lytic infection cycle or they may employ a lysogenic infection cycle in which the viral genome is incorporated into the host cell’s DNA, resulting in a latent infection. Chapter 12 key concepts

 A lytic infection cycle involves the steps of attachment, penetration, uncoating, biosynthesis, maturation, and release.  Host cell receptors are used to facilitate virus attachment and entry into host cells.  Synthesis of new viral components involves complex replication mechanisms of the viral DNA or RNA.  Intracellular trafficking and assembly of viral components in the host cell occur during the maturation stage of the viral infection cycle.

 Newly assembled viruses may be released through lysis of the host cell or by budding from the surface of the host cell.  In a one step virus growth curve for a bacteriophage the eclipse (not a single intact virion present), latent period (only intracellular virions) and rise period (virions appear extracellularly) are key phases.  The burst size is the total number of virions produced per infected cell.

A. Reverse transcriptase B. Integrase C. Polymerase D. A only E. A and B

A. Kills its host immediately B. Forms plasmids in the host C. Releases massive numbers or virus all at once D. Incorporates its nucleic acid with that of the host chromosome E. Causes host cell to become lytic.

A. Not a single intact virion is present. B. No extracellular virions are detectable. C. Release of virus progeny occurs. D. The virus adjusts to the new surronding. E. None of the above.

 11:40am – 1:20pm  Chapters 1 thru 13: Lecture, Reading, Chapter End Self Study Questions  Fifty Multiple Choice Questions = 100 points  Please bring: ◦ Scantron (form No. 882-E for the Quiz – available at no cost at the Student Bookstore) ◦ No. 2 pencil only