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Virus Multiplication

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McMaster University
Laura Parker

Virus Multiplication (Lecture 21) • Virus – consists of a cluster of genes that can replicate insides cells • Simplicity in their genetic organization  consist of DNA or RNA, not both • Obligate intracellular parasites Patterns of virion structure • Core – viral nucleic acid • Protein shell  capsid – either icosahedral or helical • Some viruses have lipoprotein envelope surrounding the capsid • Capsid – built from multiple copies of identical structural units • Icosahedron – 20 triangular faces, 12 vertices  2,3 and 5 fold symmetry Other virion proteins • Contain small amounts of proteins that play critical roles after infection • Sometimes they contain enzymes that catalyze rxns that do not normally occur in unifected animal cells, such as RNA-dependent RNA polymerase or reverse transcriptase  good targets for anti-viral therapy since not in animal cells • Other times, they are integrases, transcription factors, and proteases Classification • DNA or RNa, polarity (plus  same as mRNA, or minus), structure (linear, circular, single- or double- stranded) • Type of symmetry (helical or icosahedral) of capsid • Presence/absence of lipoprotein envelope • Presence/absence of specific virion enzymes Virus infection • One-step growth curve • Eclipse – very few infectious virus particles can be detected either inside or outside the cell during this time • Intracellular accumulation period – newly-synthesized progeny virions can be found inside but not outside the cell • Rise period – increasing numbers of progeny virions appear in the extracellular fluid • 2 ways to count viruses: o functional – apply virus to layer of cells, count plaques (pfu) o physical – count viral particles with electron microscope o particle number is usually greater than pfu number 3 basic processes common to all virus infections: • Delivery of viral genome into the cell  involves attachment of the virion to the cell and liberation of the viral nucleic acid • Synthesis of new viral genomes and virion structural proteins • Assembly and release of infectious progeny virions Attachment • Involves interaction between certain proteins on the virion surface and specific receptors on the cell surface • Receptors are normal membrane proteins or carbohydrates • Some receptors found in most cells while others are specific to certain cell types • Specificity of attachment determines the virus host range Penetration and uncoating • Viruses enter the cell by two major routes • Endocytosis – both enveloped and nonenveloped are taken into cells this way o following attachment, the virus-receptor complex migrates to coated pits and enter the cell in clathrin-coated vesicles produced by pinching off of invaginations of the cytoplasmic membrane o this is analogous to receptor mediated endocytosis of nonviral ligands o the acidic pH of the endosome triggers conformational changes that activate the fusion proteins of enveloped viruses  the resulting fusion of the viral envelope with the membrane of the endosome releases the viral nucleocapsid into the cytoplasm o uncertain how nonenveloped viruses escape from the endosome o influenza virus HA protein  sheds light on mechanism of acid-induced membrane fusion  acidic pH causes a loop segment in the HA protein to adopt an alpha helical conformation  the result of this is that a hydrophobic segment (fusogenic peptide) at the N-terminus of ha2 is moved over 100 angstroms from a buries position to the top of the HA molecules where is inserts into the membrane of the endosome. • Direct fusion o Paramyxoviruses (and a few others) enter cells by fusion of the viral envelope membrane with the cytoplasmic membrane o Process requires a specific fusion protein (F protein ) on the viral envelope. o Membrane fusion results in the delivery of a naked nucelopcapsid to cytoplasm of the cell – viral envelope glycoproteins are left embedded in the plasma membrane Synthetic phase of virus multiplication SV40 • naked icosahedral capsid surrounding the viral genome which is complexed with histones. Genome is dsDNA ~5000 bp (one of the smaller virus genomes) • 4 genes essential for virus multiplication (T antigen, VP1, VP2, VP3) • SV40 virions enter cell by endocytosis  after uncoating, the viral genome migrates into cell nucleus when multiplication takes place • 2 phases of viral genome expression o Early phase (prior to viral DNA replication) – the early region of viral genome transcribed into mRNA which directs synthesis of the T antigen (aka A protein)  T antigen accumulates in the nucleus where it performs several important functions • Induces the host cell to enter S phase so that cellular enzymes involved in DNA replication are expressed (e.g DNA pol, ligase, nucleoside kinases) • Initiates the SV40 DNA replication at the viral origin (serves as helicase) • Activates late mRNA synthesis (boundary between early and late phases) • Represses early mRNA synthesis (own synthesis) • Prevents the host cell from undergoing apoptosis  T antigen – site-specific DNA binding protein with intrinsic helicase activity • Initiation of SV40 DNA replication occurs when T antigen binds to the origin of replication and unwinds the 2 parental strands of DNA. It also recruits cellular replication proteins to the origin resulting the in the establishment of 2 replication forks. The forks proceed bidirectionally from the origin, syntheisizing in the semi-conservative mechanism. 2 daighter molecules are produced when the growing points meet roughly 180 degrees around the genome. o Late phase – at this time mRNAs transcribed from the late region of the genome appear in the cytoplasm and direct the synthesis of VP1, VP2, and VP3. These mRNAs synt
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