Transcription strategies of viruses

• Majority of DNA viruses depend on cellular RNA polymerase II, including

Parvovirus (AAV), Papovavirus (SV40), Hepadnavirus (Hapatitis B), Adenovirus (human adenovirus), Herpesvirus (herpes simplex virus)

• Retroviruses also use cellular RNA polymerase II

• The mechanisms of transcription and the signals are similar between viral and cellular genes

• Poxviruses are transcribed by viral DNA-dependent RNA polymerase and accessory viral proteins that control recognition of viral promoters

• In cells infected with DNA viruses, viral genes are expressed in a strictly defined reproducible order:

-viral enzymes and regulatory proteins are made first

-structural proteins are synthesized only after viral DNA synthesis begins

-transcription of viral genes is activated during specific periods in the infection cycle.

Several of the 12-16 subunits of these enzymes are identical

-they can bind to rNTP substrates, template DNA and product RNA

-they can catalyze phosphodiester bond formation

The transcription cycle

• Initiation- The transcriptional machinery binds to the promoter and induces local unwinding of the double-stranded DNA template

• Elongation- The transcribing complex progressively unwinds the template as it reads the DNA sequence and adds nucleotides to the 3’ end of the nascent RNA chain

• Termination- Transcription ceases when the termination signal is encountered and both the RNA transcript and the transcriptional machinery are released from the DNA template

The transcription cycle

RNA polymerase II transcriptional control elements

Mechanisms of enhancer action

• DNA looping model postulates that proteins bound to a distant enhancer interact directly with components of the transcription initiation complex, by looping out the DNA

• An enhancer noncovalently linked to a promoter via a protein bridge is functional

• Enhancer function requires close proximity to the promoter

• Enhancers do not serve as entry sites for RNA polymerase II

Initiation of transcription by RNA polymerase II

Modular organization of transcriptional activators

• Composed of different domains

-DNA binding domain

-Activation domain

• Bind to DNA as dimers

Structure of a basic leucine zipper domain bound to DNA

• Leucine zipper forms a -helical coiled coil

• DNA binding region is also -helical, but in solution it is disordered

• DNA binding induces a major conformational change

• Proteins of this class bind to DNA as dimers

f-b

Transcriptional control region of avian leukosis virus

General mechanisms of stimulation of transcription by viral proteins

• Protein A stimulates transcription of the same transcription unit (A)

or a different transcription unit (B)

Regulation of transcription by the Tat protein of HIV-1

Regulation of HIV-1 transcription by the Tat protein

• Stimulation of HIV-1 transcription by Tat requires an LTR sequence, termed the trans-activation response (TAR) sequence

• TAR element is recognized as RNA

• Tat protein binds specifically to a trinucleotide bulge in the stem of the TAR RNA stem loop structure

• Binding of Tat to this region of TAR induces local conformational rearrangement in the RNA, resulting in formation of a more compact structure

• Binding of Tat to TAR stimulates production of viral RNA as much as 100-fold

• Tat protein has little effect on initiation, it greatly improves elongation

Stimulation of transcription by HIV-1 Tat protein

• Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site

• Production of the Tat protein allows transcription complexes to synthesize full length RNA

• Binding of Tat to TAR together with the cyclin T subunit of Tak leads to stimulation of phosphorylation of the largest subunit of RNA polymerase II

• The transcriptional complexes become competent to carry out transcription

Transcriptional strategies of DNA viruses

• Viral genes are transcribed in a reproducible and precise order.

• During immediate-early and early phases viral proteins necessary for viral DNA synthesis are produced.

• Transcription of the late genes, most of which encode viral structural proteins, requires viral DNA synthesis.

• This property ensures coordinated production of the DNA genomes and structural proteins from which virus particles are assembled.

• Viral proteins and replication of viral DNA control the transition from one transcriptional stage to the next.

• Many transcription activating proteins (SV40 T antigen, HSV ICP4 protein) can repress transcription of their own genes.

Transcription of different DNA viruses

SV40

• SV 40 contains only two transcription units, early and late

• Early domain is transcribed from one strand and late domain is transcribed from another strand

• An origin serves for transcription by RNA PolII and for DNA replication

• Expression of early transcription unit leads to synthesis of large T antigen (LT)

• Expression of T antigen in the absence of viral infection leads to cell transformation and formation of tumors in animals

• LT binds to ori, regulates its own production also DNA replication

• Binding unwinds the DNA allows DNA polymerase and the initiation factors to bind to form an initiation complex

• Host specific association with -primase occurs during initiation

• LT also activates transcription of late mRNAs

• Eight transcription units encoding more than 40 viral proteins

• Transcribed in the nucleus by RNA Pol II to produce a set of early RNAs

• Upon entry of viral genome into the nucleus, E1A is transcribed.

• E1A and E1B products are oncogenes that stimulate cellular replication and induce an environment for viral DNA replication

• E1A is necessary for transcription of all viral early transcription units

• Proteins from E2 region are involved in replicating viral DNA

• Multiple splicing events that occur during processing of the adenoviral late mRNA led to the discovery of RNA splicing by Phil Sharp (1993 Nobel Prize)

• VA genes are transcribed by RNA Pol III, short RNAs are not translated

Adenovirus:

• More that 80 genes are expressed as individual transcription units

• A viral activating protein is imported into cells infected by HSV.

• This virion structural protein, VP16, is necessary for efficient transcription of viral immediate-early genes.

• VP16 resembles adenoviral E1A in performing regulatory functions

• ICP4 protein is the major transcriptional activator.

• ICP4 stimulates transcription of both early and late genes, also acts as a repressor of immediate-early gene transcription.

Herpes simplex virus: