Unit 6.1 2015 Gene Expression and Replication in Small DNA Viruses Microviridae Small DNA Viruses...

Unit 6.1 2015Unit 6.1 2015Gene Expression and Gene Expression and

Replication in Small DNA Replication in Small DNA VirusesViruses

MicroviridaeMicroviridaeSmall DNA Viruses of Animals Small DNA Viruses of Animals

(Papovaviruses)(Papovaviruses)

What do the small DNA viruses have to What do the small DNA viruses have to tell us about viral approaches to gene tell us about viral approaches to gene

expression and replication?expression and replication?What are their issues, problems and What are their issues, problems and

strategies?strategies?Are there common themes?Are there common themes?

Microviridae-PhiX174Microviridae-PhiX174

Historically important virus because… first viral genome to be sequenced (1977) + sense ss DNA circular 5386 nucleotides-very small genome genome infectious by itself model for fundamentals of DNA replication

PhiX174 –among smallest DNA PhiX174 –among smallest DNA virusesviruses

T=1 CapsidT=1 CapsidSs + DNA circleSs + DNA circleConverted to ds immediately upon entry = “RF”Converted to ds immediately upon entry = “RF”11 gene products identified11 gene products identified

Attachment and Penetration of PhiX174

•Attaches via vertex & pilot protein H binds to a specific lipopolysaccharide in outer cell membrane of its host, E. coli or S. typhimurium•The pilot protein leads the circular genome into the cell by an energy-dependent mechanism•The genome attaches to the inner bacterial membrane in preparation for DNA replication and RF formation.

Gram Negative Cell Wall

11 Gene Products11 Gene Products A: DNA replication (513)A: DNA replication (513) A*: inhibit host replication????? (341)A*: inhibit host replication????? (341) B: capsid assembly protein (internal) (120)B: capsid assembly protein (internal) (120) C: regulation of replication (86)C: regulation of replication (86) D: capsid assembly protein (external) (152)D: capsid assembly protein (external) (152) E: lysis (91)E: lysis (91) F: major capsid protein (427)F: major capsid protein (427) G: spike protein (175)G: spike protein (175) H: pilot protein (328)H: pilot protein (328) J: DNA binding protein(capsid assembly) (38)J: DNA binding protein(capsid assembly) (38) K: Burst size regulation???? (56)K: Burst size regulation???? (56)

Genome is very small for 11 gene Genome is very small for 11 gene productsproducts

Genome sequencing Genome sequencing reveals answerreveals answer

Seven genes overlap at Seven genes overlap at least one other geneleast one other gene

Gene for A* overlaps 3 Gene for A* overlaps 3 other genesother genes

Frameshift or use of Frameshift or use of alternate AUG codonalternate AUG codon

Non-capsid genes Non-capsid genes involvedinvolved

Rolling Circle Replication-”sigma”Rolling Circle Replication-”sigma”

The RF is made by The RF is made by host enzymeshost enzymes(-) strand template (-) strand template for (+)for (+)Multiple copies of Multiple copies of (+) made in a chain(+) made in a chainSingle genomes cut Single genomes cut outout

(+) is nicked by A (+) is nicked by A proteinprotein

Small genome = Dependence on Small genome = Dependence on Host Host

RF is produced by action of host RF is produced by action of host DdDp enzymesDdDp enzymes

Progeny genomes synthesized by Progeny genomes synthesized by rolling circle mechanism (host and rolling circle mechanism (host and

viral protein A)viral protein A)Transcription by host DdRp Transcription by host DdRp

enzymeenzyme