Virus Replication. Virus-Infected Cells. Virus Replication. Microbiology Lecture 6. Virus Replication in Tissue Culture.

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1 Virus Replication Microbiology Lecture 6 Professor T.J. Foster Virus Replication Can causes many different changes to host cells Cytopathic Effects (CPE) Altered shape Lysis Membrane fusion (giant cell formation) Inclusion bodies Programmed cell death Detachment Virus Replication in Tissue Culture. Lysis Virus-Infected Cells Infection of monolayer of tissue culture cells. Plaques result from infection of single cell by single virus causing LYSIS. Serial dilution of stock allows virus concentration to be counted as plaque forming units (pfu) Monolayer of epithelial cells in tissue culture Rhinovirus-infected Cells ALTERED SHAPE 1

2 Negri body Inclusion Body Formation. Rabies Virus Budding virus Syncytium [Giant Cell] Formation Abundant nucleoprotein Healthy neurons Negri Body in infected neuron Measles Virus Programmed Cell Death Apoptosis Virus Multiplication. General Principles Illustrated with Enveloped, Single Negative Stranded RNA Virus Normal brain-derived glioma cells Apoptosis caused by West Nile Virus. Condensation of nucleus. Membrane dissolution. Blebbing Baltimore Group 5 Eg. Influenza Virus 2

3 Envelope glycoproteins Capsid protein Gene expression Bind to specific viral receptor molecules on certain types of cells from particular animal hosts Determines virus tissue and host TROPISM Retrovirus Envelope Glycoprotein HIV binds CD4. CXCR4 or CCR5 are co-receptors Host receptor is CD4 on T lymphocytes. CD4 is important in antigen presentation (lecture 8) 3

4 Coronaviridae Human Coronavirus SARS virus Orthomyxoviridae Influenza viruses Adenoviruses Envelope spike protein binds to sialic (neuraminic) acid on surface of mammalian cells Haemagglutinin binds to sialic (neuraminic acid) on surface of mammalian cells. Neuraminidase facilitates release. Target for anti-influenza drug (Tamiflu). CAR = Coxackie & Adenovirus Receptor (for tip of spike) Host cell INTEGRIN binds specifically to capsomer protein on capsid surface Virus Multiplication. General Principles Illustrated with Enveloped, Single Negative Stranded RNA Virus Baltimore Group 5 Eg. Influenza Virus Gene expression 4

5 - Entry Energy-dependent Cells must be metabolically active Three mechanisms 1. Translocation of whole particle 2. Receptor-mediated endocytosis. Taken into vacuoles. Influenza virus [enveloped] & adenovirus [naked] 3. Fusion. Enveloped virus. Membrane of envelope fuses with cytoplasmic membrane of host cell. Retrovirus HIV Virus Membrane Fusion Influenza Virus Endocytosis Gene expression UNCOATING RELEASE OF NUCLEIC ACID FROM CAPSID At plasma membrane In cytoplasm Poliovirus In endosome [triggered by lower ph] Influenza virus Herpes virus replicates in nucleus Nucleocapsid transported to nucleus. Capsid stripped off during passage of genome through pore in nuclear membrane 5

6 Nucleic Acid Replication Gene expression Baltimore 5 -ss RNA Replication -ssrna. ds RNA (+/-) acts as template for genome copies (-ssrna) Great Diversity of (Baltimore Classification Scheme) Cellular enzymes replicate viral genome Parvovirus (ssdna) : rare Virus-encoded proteins replicate viral genome [most common] DNA Structure Baltimore Classification Group Genome Information Example 1 ds DNA dsdna mrna Herpes simplex virus 2 ss DNA ssdna dsdna mrna Parvovirus 3 ds RNA dsrna mrna Reovirus 4 + ss RNA Serves as mrna 5 - ss RNA mrna template dsrna +ssrna (mrna) Enterovirus dsrna -ssrna mrna Influenza A 6 ssrna ssrna dsdna mrna Retrovirus Double stranded (ds)dna duplex Ribonucleic acid can also form a ds duplex 7 Nicked ds DNA nicked ds intact ds mrna DNA DNA RNA Hepatitis B virus 6

7 Synthesis of Viral Proteins Capsid proteins synthesized in cytoplasm directed by viral mrna Envelope proteins synthesized in endoplasmic reticulum and transported to cytoplasmic membrane Protein synthesis Capsid Assembly Capsomer proteins assemble around viral genome. Capsid assembles [self assembly] In cytoplasm picornavirus, poxvirus Protein synthesis In nucleus adenovirus, herpes simplex virus 7

8 Non-enveloped viruses Cell lysis Enveloped viruses Budding May or may not kill cell Controlled by virus Virus membrane glycoproteins incorporated Gene expression Herpes Simplex Virus Enveloped DS DNA Virus (Baltimore 1) Nucleocapsid transported along microtubules to nucleus. Docks over nuclear pore. DNA injected into nucleus, capsid proteins remain in cytoplasm. Transcription Early infecting virus DNA template. Promote virus DNA Late replicated virus DNA template. Capsid proteins migrate to nucleus. Envelope glycoproteins Synthesized in ER. Migrate to nuclear membrane Nucleocapsid buds through nuclear membrane acquiring envelope 8

9 Nucleocapsid transported along microtubules to nucleus. Docks over nuclear pore. DNA injected into nucleus Transcription Early infecting virus, DNA template. Promote virus DNA Late replicated virus DNA template. Capsid proteins migrate to nucleus. Envelope glycoproteins Synthesized in ER. Migrate to nuclear membrane Nucleocapsid buds through nuclear membrane acquiring envelope Passes through ER and Golgi Exocytosis through Cytoplasmic membrane Retrovirus Enveloped virus binds to CD4+ cells Fusion of virus envelope with host cell membrane of virus RNA & proteins (integrase, reverse transcriptase, ribonuclease) Reverse transcriptase copies virus RNA into cdna Ribonuclease H destroys RNA strand RT copies DNA strand to make DS DNA Retrovirus DS DNA migrates to Nucleus Integrase splices virus DNA into host chromosome > provirus Transcription (i) New viral genomes. (ii) mrna translated into virus capsid proteins and associated proteins In ER viral envelope proteins. Migrate via Golgi to cell membrane 9

10 Retrovirus Herpes Virus Replication Retrovirus Replication For an animated version of Herpes Virus For an animated version of Retrovirus Virus by Budding Acquiring envelope Antiviral Drugs Difficult to identify drug targets Viruses employ host enzymes/ribosomes Influenza virus neuraminidase HIV retrovirus encoded enzymes Reverse transcriptase Viral protease [processing viral capsid proteins prior to assembly] The focus of lectures 5 & 6 is the structure and of viruses. The structure of several important types of viral capsids are described - the simple polyhedral capsid and the helical elongated capsid. Both are composed of capsomer proteins that aggregate around the viral nucleic acid genome, be it RNA or DNA. Some viruses have more complex capsid structures and are composed of several different capsid structural proteins. Some viruses that infect mammalian cells have an additional membranous ENVELOPE surrounding the capsid. This is derived from the membrane of the infected host cell as the virus escapes following intracellular. The envelope has virus-expressed glycoproteins embedded in it. These are also expressed from viral genes during and render the envelope recognisable as foreign. Bacteria can be infected by viruses called bacteriophages. Some phage have bizarre tail structures that allow them to attach onto the surface of a cell and to inject their DNA into the cytoplasm. The head and tail proteins of phages remain OUTSIDE the cell during the process of infection, in contrast to viruses that infect mammalian cells where the whole virus is internalized. The sequence of events that occur within virus-infected cells is considered. This is an opportunity to revise your knowledge of DNA, transcription and translation. The Baltimore classification scheme gives 7 classes of viruses based on the nucleic acid composition of their genomes and the process of and gene expression. Multiplication of viruses within host cells is considered under several general headings: attachment, uptake, uncoating, nucleic acid, viral gene expression and protein synthesis, assembly and release. Several examples are considered, an ssrna virus that replicates in the nucleus, a ssdna virus that is converted to dsdna and integrates into the hosts genome (retrovirus) and dsdna virus that replicates in the nucleus (herpes virus). Retrovirus. The virus enters the cell by endocytosis. The capsid proteins are removed. The virus ssrna genome is copied into dsdna by an enzyme called reverse transcriptase, the signature of the retrovirus. The enzyme is present within the capsid and is activated in the cytosol of the infected cell. The DNA is converted into the double stranded form and migrates to the nucleus where it integrates into the chromosome forming a provirus. The viral DNA is then copied back into genomic RNA and mrna which is translated into viral proteins, including capsid proteins, more reverse transcriptase and the envelope glycoprotein. The virus emerges from the infected cell by budding. HIV specifically infects CD4+ T cells (lecture 8) leading to immunodeficiency. Vaccines for virus infections. Many vaccines are currently used to prevent virus infections (lecture 8). These are live-attenuated or inactivated virus particles. Note, a vaccine for HIV has proved impossible to develope because of high frequency antigenic variation, particularly in the surface glycoprotein. Error-prone by reverse transcriptase allows a high frequency of mutation Treatment of viral infections. Traditional antibiotics designed to treat bacterial infections will have no effect on a viral infection. Bacteria are living prokaryotic cells while viruses grow and multiply within host human cells (eukaryotic). Some viral polymerases have been targeted with specific inhibitors, also the HIV-specified protease, and the influenza virus neuraminidase. Reading material: Campbell has an adequate chapter 10