Gene Expression and Replication in Medium DNA Viruses Phage Lambda Adenovirus

What can the medium sized viruses do that the small ones can not What can the medium sized viruses do that the small ones can not? Adenovirus: more sophisticated gene expression strategies Lambda: two pathways for infection

Phage Lambda Bacteriophage isolated from human gut-Andre Lvov? dsDNA, 48.5 kbp Linear, sticky ends “cos” Complex capsid Unusual biological properties noticed Model system from 1950’s Cloning vector

Evergreen State-Olympia Mansfield State (Ohio) The Evergreen State College Phage Lab has been a center for undergraduate research at Evergreen since Betty Kutter came here in 1972, one year after the college opened. Today, there are generally 10-15 students involved in work in the lab under the direction of Kutter and faculty colleague Andrew Brabban. We are focusing particularly on phage ecology, with the aims of understanding the infection process under anaerobic and other conditions better reflecting those in the natural environment, as well as on interactions between phages of different families during simultaneous infection and the building of cocktails for potential therapeutic applications. Dr. Elizabeth Kutter: KutterB@evergreen.edu Dr. Andrew Brabban: BrabbanA@evergreen.edu Evergreen State-Olympia Mansfield State (Ohio) Bacteriophage Therapy Inst-Tbilisi Bacteriophage therapy was used broadly in the Soviet Union, particularly the Republic of Georgia, which has been the global center of phage expertise for over 80 years.

Lambda Entry and Uncoating Non-contractile Lambda J protein uses Lam B as receptor DNA enters through mannose permease complex

Lambda genetic map Shortly after entry the linear DNA forms a circle Cos sites are complementary with one another Assembles functional regions

Lambda’s two developmental pathways In some cases lambda follows the “usual” pathway in which the host cell is killed straightaway-the “lytic” pathway. In other cases lambda DNA inserts into the bacterial chromosome to form a “prophage”. The prophage remains in the chromosome as a passenger until the host cell is stressed or “induced”. Then it lyses the host cell to release new phage. The latter pathway is called the “lysogenic” or “prophage” pathway. (temperate, vegetative)

Lysis and Lysogeny for Phage Lambda What are the advantages of lysogeny for lambda? How does lambda make its decision for lysis or lysogeny?

Phage lambda regulatory region Key region regulates developmental decision P = promoter O = operator T = terminator

Operon Model Shown for lactose operon Repressor protein binds to operator sequence: No mRNA so gene is off! Inducer causes shape change in repressor Repressor drops off operator mRNA produced so gene is on

Gene Expression Immediately after infection, N and cro are transcribed by host RdRp (“immediate early genes”) N is an antiterminator- it allows transcription and expression of cIII, cII, O, Q (“early genes”). Cro is a repressor. It can block transcription from Pr and Pl. N says “go”, cro says “stop”

Lytic pathway If cro builds up quickly enough it prevents further synthesis of N and of cro itself. Q allows expression of “late” genes that encode for lytic functions.

Establishment of Lysogeny If cII builds up fast enough it activates the transcription of cI and also some integration functions. cI is called the lambda repressor. It shuts off all lambda genes except itself and allows lambda DNA to integrate into the host chromosome.

Lysis and Lysogeny Compete If cro “wins” then late genes are expressed and lysis follows. If N, cII, cIII win then the lambda repressor is expressed from cI and lysogeny follows. This medium-sized virus is complex enough for two developmental options.

Post Script:Lambda repressor binding Binds to operator as a homodimer Helix-turn-helix protein Up to 3 dimers can bind Binding is cooperative

Cooperative repressor binding produces steep response curve-”all or nothing” response R E P R E S S I O N REPRESSOR CONCENTRATION

When enough repressor is around is turns off its own synthesis When repressor levels drop-more is made Induction involves destruction of repressor Superinfection immunity

Lambda DNA Replication Lambda uses a combination of host and viral proteins. Theta strategy for first few rounds of replication. Then shifts to rolling circle. Long tail converted to ds concatemer. Cleavage occurs at cos sites during packaging.