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

y 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

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

o o o

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

o o o o

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

o o o

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

o o

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 LTRs 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