DNA VIRUSES

DNA Viruses

• Except for poxviruses, transcription occurs in the nucleus and translation in the cytoplasm.

• Generally, the primary transcripts, generated by RNA polymerase II, are larger than the mRNAs found on ribosomes, and in some cases, as much as 30% of the transcribed RNA remains untranslated in the nucleus.

• The viral messengers, however, like those of animal cells, are monocistronic. • Transcription has a temporal organization, with most DNA viruses only a

small fraction of the genome is transcribed into early messengers. • The synthesis of early proteins is the key initial step in viral DNA replication. • After DNA synthesis, the remainder of the genome is transcribed into late

messengers. • The complex viruses have immediate early genes, which are expressed in the

presence of inhibitors of protein synthesis, and delayed early genes, which require protein synthesis for expression.

• Regulation is carried out by proteins present in the virions, or specified by viral or cellular genes, interacting with regulatory sequences at the 5' end of the genes.

DNA (genome) replication strategies similar in all and similar to host

• ssDNA becomes dsDNA• 5’ to 3’ synthesis; need for

primer• Variety of enzymes of host

or viral origin : DNA polymerase (proofreading), helicases, ss binding proteins, ligases

• In nucleus except for poxvirusesPhage T4 replisome

Replication Challenges for DNAViruses

• Access to nucleus• Competing for

nucleotides• Cell cycle control in

eucaryotes - S phase dependent materials

Transcriptional/translational challenges

• Access to RNA polymerase

• Monogenic expression in eukaryotes

• Temporal control of gene expression

• Competition with host for ribosomes

Bacteriophages: T4

• Linear dsDNA - ~ 1.2 x 10^8 d (>280 genes)

• Terminally redundant ends enable circularization

What affect does T4 infection have on macromolecular synthesis in the cell?

• How would you measure DNA synthesis? RNA synthesis? Protein synthesis?

• How can you distinguish between phage and host DNA synthesis?

• How can you distinguish between phage and host RNA synthesis?

RNA

protein

DNA

Rel

conc

time0

RNA production in cell

• Temporal control of transcription– Immediate early (IE): will occur in presence of

ps inhibitor What RNA-P is used?– Delayed early (DE) - needs protein synthesis

and before DNA replication– Late - after DNA replication begins - structural

proteins

T4 changes host RNA-P

• RNA-P - 4 subunits plus sigma factor

• IE uses host enzyme but at promotors that differ from E. coli (high affinity)

• IE gene products – modifies (ADP

ribosylation) RNA-P to recognize DE promotors

– Antitermination– Nucleases (host DNA and

tRNA)– Membrane repair

• DE further changes to RNA-P– Antisigma factor (ASiA)– Activator proteins– Phage tRNAs– Nucleotide metabolism– DNA replication

• Late requires different sigma factors

T4 genome - also 127 ORFs of unknown fucntion

Gene function % of known genome functions

Metabolic, essential (22)

15

Metabolic, unessential (60)

39

Structural (34) 27

Assembly, nonstructural (19)

10

T7 control

• Linear dsDNA– ~ 25 x 10^6d

• Unique with TR - how is this formed?

• Genes are in order of entry on chromosome

T7 promotors differ

• IE - host polymerase• Creation of a new

polymerase/inactivation of host polymerase

• T7 polymerase promoter often used in gene cloning for control of expression

Papovaviruses• Papilloma/Polyoma/

Vacuolating agent• Bidirectional replication

from single ori (similar to Bacteria)

• Early to late strategies– T ags in SV40 enhance

first and then suppresses early;

– E ag in BPV is an enhancer for late genes

– Mutations in T or Eag/transition lead to tumors

How do DNA Viruses Get cells out of G1 and into S phase

• Inactivate Rb/p53 - cell cycle regulators

• SV40 uses T ag against p53

• p53 inactivation probably stops apoptosis

• Multiple functions for T ag increases genome potential

HPV Transcription using host RNA-P

• Multiple promotors some with overlapping reading frames

• Alternative splicing - more genes for your genome

Adenovirus - 5’protein primer

• Linear dsDNA– 20-30 x 10^6 d

• Terminal protein linked to 5’nucleotide

• Sequential replication from linear DNA

• No Okazaki fragments

This is now a template

Inverted terminal repeats

Adenovirus - transcription

• Monogenic proteins with individual promotors

• Uses host RNA-P• Multliple splicing of

mRNA yields different proteins

• E1A is IE gene- activates at other E promotors

DNA replication

Poxvirus: DNA with a complex morphology

• Large genomes - 130 n- 240 x 10^6d• Denatured genome is ss circle • Replicates in cytoplasm• Brings in RNA-P; mRNA is capped • Makes all replicating enzymes

Herpes Simplex Virus• Tegument - ~ 18 proteins• Access to nucleus

–TIF (VP16 /UL48 ) trans inducing factor

• binds with host factors to begin transcription

• 500 - 1000 copies/virion• Determines tissue

tropism–VHS (UL41) degrades

preexisting mRNA but is stopped so virus can work

Temporal expression of genes

Alpha and Beta proteins

• Beta• DNA replication

(polymerae,binding proteins, helicase/primase)

• Thymidine kinase• DNA repair proteins• Turn on Gamma/off Alpha

• Gamma• Structural proteins• Tegument proteins

• Alpha • ICP27 - blocks host RNA

splicing• Immune escape (MHC1

downregulation)• Turn on Beta genes

Herpes virus supplies all DNA machinery

• No need for cell to be in S phase

• Model for replication–Rolling circle

leads to concatemers

Thymidine kinase and Ribonucleotide reductase are early proteins

• Needed for virulence but not in cell culture WHY?

• TK needed to activate acyclovir

• DNA polymerase - target of acyclovir

• Many proteins have some cellular homolog - stolen genes?– Stress response gene -

counter stress of viral infection?

Packaging of Herpesviruses

Host cell defense?

• Prevention of translational initiation is a commonly utilized mechanism of cellular anti-viral defence.

• Strategies have been adopted by viruses to overcome host cell attempts to terminate protein translation in the face of infection.

• The mechanisms utilized by viruses such as HSV, are known in some detail and the host cell enzyme PKR is a central mediator in these mechanisms.

• PKR is normally present in cells in a latent form. • It is induced by interferon following virus infection, PKR is activated

via autophosphorylation. • Activated PKR can phosphorylate the a subunit of the initiation factor

eIF-2 (eIF2a), which generally results in prevention of translational initiation and thus halts protein synthesis.

HSV -1 ICP

• Herpes simplex virus type 1 (HSV-1) infected cell protein• (ICP) 34.5 gene null mutants exhibit severely attenuated• replication in animal models of HSV pathogenesis, but

replicate as well as wild-type HSV in many malignant cells in vitro and in vivo

• Capitalizing on this selective lytic replication, it has been used to successfully treat brain tumours, including melanoma, intraperitoneal human mesothelioma and subcutaneous human melanoma, in various immunodeficient and immunocompetent mouse models.

Protection from host are early products

• Prevention of apoptosis

• Use mutants and see affects (ICP = infected cell protein)

• Cisplatin is apo inducer (+ control)

apoptosis

wt cisplatin ICP-