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VIROLOGY MB-29: Viral Structure and Classification How vaccines produce immunity  Induce neutralizing antibodies + cell immunity  kills

pathogen  Killed virus (Salk) vs live attenuated vaccine (Sabin) vs Subunit vaccines (HPV) What are viruses?  Only contain nucleic acids + proteins  Obligatory intracellular  Potentially pathogenic (there are viruses that cause no harm!)  RNA or DNA (NOT both)  Replicate from their genetic material Virion – structural proteins  RNA or DNA  Single or double stranded  Linear or circular  One or several pieces  Plus (mRNA) or minus strand Structure  Viral shell (capsid)  Helical vs cubic symmetry

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Cubic o o o o

Icosahedron: 20 triangle faces, 12 corners,  60 equivalent subunits Capsomer: pentamers (5-coordinated culsters) Hexamers: 6-coordinated clusters C = (T-1) x 10 + 12  C = # capsomers  T = # subtriangles (triangulation #)

Nonviron – nonstructural proteins  Enzymes for synth of nucleic acids Naked virus: just capsid + nucleic acid Enveloped virus: includes a bimolecular membranous coat

Nomenclature  Horne-Lwoff-Tournier – based on 3 classifications o RNA vs DNA o Helical vs cubic o Naked vs enveloped o  8 classes  Baltimore system o Classes I-VII o Based on starting nucleic acid  how it becomes mRNA  Ex: dsDNA mRNA  ssDNA  dsDNA  mRNA Virology assays o ELISA o darker color = more virus

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o Electron microscopy (research) PCR o Determines copy # of viral genome in blood sample Plaque assay o Research use o Empty areas = plaque (one Plaque Forming Unit – PFU = 1 viral particle)

MB-30: RNA viruses 11.8.11 Important RNA viruses o Rhinovirus – common cold o Aphthoviruses – from livestock

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Alphaviruses – equine encephalitis Rubellavirus – german measles Rotavirus – acute diarrhea in infants Coronavirus - SARS

Arboviruses: arthropod-borne-viruses o Flaviviruses (yellow fever virus, St Louis encephalitis, Japanese B encephalitis, West Nile virus) Viral replication (ex: poliovirus) o Poliovirus: 180 subunits, naked icosahedral ssRNA(+) virus o Adsorption o Electrostatic interaction between divalent cations o Tissue tropism – certain tissues display cell receptors for poliovirus o Penetration o Inject genome into host cell o Polio – uptake of VP4 capsid protein is important  Energy stored in viral capsid o Uncoating o RNA liberated from capsid o In poliovirus, only 2% adsorb, penetrate, and are uncoated successfully o Eclipse o When viral genome is liberated in cytoplasm & it has not yet started viral replication, and no progeny are made yet o Appears to have ‘disappeared’ o Nucleic acids are in free form & are susceptible to enzymatic degradation o Maturation & Release o Virus replicates & grows intracellulary o Then released all at once as free virus o Biology of poliovirus o RNA codes for:  Inhibitors of cellular RNA & protein synthesis  RNA-dependent RNA polymerase  Virion structural proteins  Potease (virus assembly & maturation) o +ssRNA  complementary strand (negative)  mRNA

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Polycistronic  RNA contains info for several proteins Assembly of virion

MB-31: INTRO TO RETROVIRUSES 11.10.11 Retrovirus structure o Enveloped, dsRNA o Contains reverse transcriptase, integrase, and protease o 3 polyproteins (Gag, Pol, Env)  8 final proteins

Life cycle o dsRNA  dsDNA via reverse transcriptase o host tRNA helps prime for reverse transcription o Initial RNA degraded by RNAseH activity (part of reverse transcriptase) o Second DNA strand copied at ‘PPT site’ o Results in dsDNA DNA integrated to genome (via integrase) o Makes sticky ends by cutting out 2 bases on each end o Makes cuts in host DNA o Viral DNA is ligated o Tends to be in chromosomes with more activity / replication Viral genome replicates Viral particles assemble, bud from membrane, and go to infect other cells


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o o HIV enters new cells o Gp120 binds CD4  conformation change  Binds CCR5  conformational change 

Genome o Gag = structural proteins (MA, CA, NC) o Pol = enzymes in particles (protease, reverse transcriptase, integrase) o Env = fusion protein (what is on outside of envelope that binds to receptor) o LTR’s o 5’ = promoter  many binding sites (ex: NF_kB) o 3’ = polyadenylation o Gene expression o Problem: only 1 promoter so can only make 1 primary transcript / mRNA o Solution: ribosomal frameshifting: ‘slippage’ causes translation to continue past the stop codon after Gag  Gag-Pol  Splicing  Env (and even more complex in HIV) o Protease cuts up these pieces into smaller proteins Pharmacology – HIV Drugs o Some people lack CCR5 receptor (CCR5delta32)  highly resistant to HIV


o Can still bind to CXCR4 o Drugs try to inhibit CCR5 (Maraviroc) o Stem cell transplantation with CCR5delta32 stem cells  Berlin Patient cured Protease inhibitors are the best antiretroviral drugs

Retroviruses cause cancer o Switch on host oncogene o o ‘bad luck integration event’

retrovirus acquires cellular oncogene o proto oncogene incorporated into viral genome HTLV-1 has viral protein ‘Tax’ which transforms cells o Human T Cell Leukemia Virus o Transmitted by blood transfusion, most common in SE Asia & Caribbean


Retroviruses as gene therapy o Attempt to transfer genes into stem cells, in diseases like SCID, sickle cell anemia, thalassemia, etc o Downside = chance of insertional oncogenesis  turns on proto-oncogene LMO-2  Leukemia