General Virology

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General Virology

 

VIRUS STRUCTURE  

Virion vs virus • Virion is the infectious  particle – composed of nucleic  acid, protein capsid,  +/­ envelope – may be extracellular or  intracellular • Virus is any stage of  infection  

 

How do we know that NA is genetic  material?

Hershey­Chase

 

Fraenkel­Conrat  Experiment          

TRANSFECTION    EXPTS

TRANSFECTION FAILS FOR  SOME VIRUSES WHY?

 

 

Capsid Functions – Protection of NA – Attachment for naked  viruses – Enzyme • Helical vs Icosahedral  Symmetry ­ Why do most  viruses look alike? • Tobacco mosaic virus is a  ssRNA virus composed of  6000 nucleotides. The capsid  is made of 2100 copies of a  single protein subunit that  contain 158 amino acids.  Calculate the percentage of  the genome that is used for  structure.  •

 

 

How do helical viruses differ? • Helical­ one axis of symmetry  down center • Multiple structural units

 

 

Icosahedral symmetry • 20 identical equilateral  triangles • Structural units on faces  to give morphological  capsomers – Pentons (5 fold axis of  symmetry) – Hexons • 3 fold through face • 2 fold through edge  

How do spherical  viruses differ?

 

Envelope

• Attachment • Entry • Assembly­ matrix  proteins • Release • Proteins are viral • Lipids are host • Rare in plants or bacteria ­  why?

• If the membrane envelope is  destroyed, the virus becomes  noninfectious. Why?  

 

Herpesvirus complexity • • •



 

 

Tegument proteins ­ 12/84 viral  proteins in HSV Potential role? Virion mRNA   – DNAase virion nucleic  acids – RT­PCR  – probe genome array Potential role?

Genome ­ DNA or RNA Genome ­  How do we  experimentally show that  DNA or RNA is the virus  genetic material?

 

• strandedness - (single) (double) • linear or circular, partial double stranded circle • number (single, segmented, multicomponent)  

RNA Genomes • sense (positive-sense, negative-sense, ambisense) • presence or absence of 5'-terminal cap or 5'-covalently-linked protein • presence or absence of 3'-terminal poly (A) tract • Retroviruses - replication strategy  

 

Some viruses have high degree of secondary  structure • Poliovirus ­ 5’ internal  ribosome entry site (IRES)

Guest et al. 2004. J. Virol. 78: 11097.

 

 

SARS/coronaviruses have conserved 3’region • • •





Robertson et al. 2005. PLOsBiology:3.

 

 

SARS s2m in red a ­ green = 530 loop of 16S RNA Similar binding properties: – b ­ blue = S12 – magenta = IF1 Possible role for s2m – Hijacks protein synthesis from  cell(binding cell factors) – Needed to bind to similar viral  protein for transcription Potential drug target in red tunnel

DNA Viruses may be large genomes

 

 



PolyDNAvirus (PDV) ­ contain  many DNA segments



Mimivirus ­ larger than small  bacteria

Host­induced modification • Viral property that varies  depending on the host  • Phage DNA hydroxymethyl  cytosine (HMC) replaces C  – Viral enzymes: C to  HMC – Viral DNA polymerase:  adds HMC not C – What is advantage of  HMC? • Glucose is attached to HMC – Host enzyme needed to  prepare glucose – Protects against host    nuclease

 

• What would happen if  virus without glucose  enters host with RE? • What would happen if  virus with glucose  enters host w/o  enzyme to create  UDP­ glucose?

Host enzyme makes

 

 

Proteins • structural proteins  • non­structural virion  proteins – transcriptase,  – protease – integrase

 

 

How to identify virion proteins • • •

 

 

Purify KSHV virions Run on SDS PAGE Excise bands, digest ­ get  sequence and compare to  database

Chemical synthesis of poliovirus: What are  the implications? • Small genome positive strand RNA ­ sequence known • Synthesized small DNA segments (~ 69 nucleotides) with  overlapping complementary segments • Added a T7 phage promotor to DNA  • Used DNA to make genome RNA in HeLa cell lysate with  T7 polymerase • Results: How do you show success?

 

 

QuickTimeª and a TIFF (Uncompressed) decompressor are needed to see this picture.

 

 

 

 

International Congress on Taxonomy of Viruses  http://www.ncbi.nlm.nih.gov/ICTV/ • Morphology – virion size – enveloped or naked  nucleocapsid – capsid symmetry and  structure

 

• Genome characteristics • Replication strategy • Antigenic Properties

 

Baltimore classification

 

 

WHY TRANSFECTION FAILS

 

 

ONE STEP GROWTH CURVE • •

• • •

 

1939­ Ellis and Delbruck:  Infection with a high multiplicity  of infection (MOI): ratio of virus  to host cell – Simultaneous infection  – Single replication cycle Sample at time intervals by plaque  count for plaque­forming units  (PFU), Identification of latent phase Determination of burst size/viral  yield

 

Measuring Intracellular Events • Sample at time intervals  after lysing cells (1952 ­  Doermann) – Chloroform – Lysis from without • Identification of eclipse  and maturation phases

 

Maturation phase

 

 

 

• Strategy of replication – Lytic – Temperate

 

 

Biologic Properties • natural host range • mode of transmission in nature • vector relationships • geographic distribution • pathogenicity, association with disease • tissue tropisms, pathology, histopathology  

 

How can you identify these viruses?

 

 

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