Dna Viruses

DNA VIRUSES

DNA replication

Things to Think: Hard competition: Number of promoters in the host >10000; and the virus? Strong promoters for the IE stage! What helps?

Enhancers!!! Host Origin of Replication: Replication once in a life time (every cell cycle) Virus Origin of Replication: Replication Many times

Tag=large T antigen (Viral) RPA=replication protein A (host) Pol=DNA Polymerase (host) Primase (host)

Virus Structure • Icoshedral – 20 surfaces – 12 vertices

• 13% DNA • 87% Protein • NO LIPID

pTP=preTerminal protein (viral) Pol=DNA polymerase (viral) b.s=binding sight T.F.=transcription factor (host) DBP=DNA binding protein (host)

Terminal protein to initiate the replication

Terminal protein-Ser-dCMP-3’-OH Bind to 5’-end of DNA to initiate the DNA replication

The mechanism of strand displacement

•Viral DNA replication requires at least ONE viral Protein (EARLY protein) •Viral replication cannot start until a sufficient concentration of this protein has been achieved in the infected cell •A viral replicating unit is a REPLICON •The efficiency of replication depends on the functional identification of the origins

Transformation by RNA viruses  The transformation gene originate from the host - cellular origin  Integration to chromosome results in transforming event THE TRANSFORMATION EVENT IS NOT ESSENTIAL TO VIRAL REPLICATION

Transformation by DNA viruses  Transformation is connected directly to the replication of the virus  Transforming genes originate from the virus and are essential for its replication  Productive replication>>>cell death  When: The cell is non-permissive The virus is mutated REPLICATION OF THE VIRUS IS BLOCKED TRANSFORMATION EVENT WILL TAKE PLACE

Viral Cycle Early phase ‫הכנה לסינתזת‬ DNA DNA syntheses Viral mutations Non-permissive cell

Transformation

Late phase

‫יצור חלבוני מבנה‬ ‫התרבות נגיף‬

Cell Death

The target of the viral transforming proteins >>>> is the cell cycle

Why do viruses require cells is S phase?  Induction of essential substrates (that are in rate limiting) for replication: Examples: Ribonucleotide reductase Deoxynucleotides biosynthesis

What is a tumor suppressor • Tumor suppressors are proteins that inhibit cell growth in a regulated fashion to prevent abnormal growth (They are the brakes for the cell cycle) – pRB -inhibits the cell cycle – P53 -is the most commonly mutated gene in cancers (it connects DNA damage, apoptosis and cell cycle through multiple interactions)

p53

Papillomaviruses

HPV genes Only one of the strands is transcribed

• • • • • • • • • • •

Gene: Function: E1 DNA-dependent ATPase, ATP dependent helicase: allow unwinding of the viral genome and act as an elongation factor for DNA replication. E2 Responsible for recognition and binding of origin of replication. Exists in two forms: E3 ??? E4 Late Expression: C terminal binds intermediate filament, allowing release of virus-like particles. Also involved in transformation of host cell by deregulation of host cell mitogenic signalling pathway. E5 Obstruction of growth suppression mechanisms: e.g EGF receptor; E6 Transformation of host cell by binding p53 tumour suppressor protein. E7 Transforming protein, binds to pRB/p107. E8 ??? L1 Major capsid protein: can form virus-like particles. L2 Minor capsid protein: possible DNA packaging protein.

Gene expression

Adenovirus

Tri-partite leader

ADENOVIRUS GENE EXPRESSION

[Tri-partite leader]

MAJOR LATE PROMOTER (MLP)

Papillomavirus

HPV genes Only one of the strands is transcribed

• • • • • • • • • • •

Gene: Function: E1 DNA-dependent ATPase, ATP dependent helicase: allow unwinding of the viral genome and act as an elongation factor for DNA replication. E2 Responsible for recognition and binding of origin of replication. Exists in two forms: E3 ??? E4 Late Expression: C terminal binds intermediate filament, allowing release of virus-like particles. Also involved in transformation of host cell by deregulation of host cell mitogenic signaling pathway. E5 Obstruction of growth suppression mechanisms: e.g EGF receptor; E6 Transformation of host cell by binding p53 tumor suppressor protein. E7 Transforming protein, binds to pRB/p107. E8 ??? L1 Major capsid protein: can form virus-like particles. L2 Minor capsid protein: possible DNA packaging protein.

Adeno and HPV and cancer Only some adenovirus serotypes and papillomavirus Types are oncogenic. Most are either non or weakly transforming

High risk HPV strains

Oncogenicity potential is determined by conserved sequences (CR1,2,3) within the early proteins

Domains

mRNAs E1A

E1B

The molecular basis For E1A activities

DNA viruses use similar mechanisms to determine the fate of the infection (apoptosis/cell cycle arrest/cancer)

Small sequence differences may be significant

Low risk

high risk

G=Gly

D=Asp

The ability of early proteins CR regions to bind or degrade cellular tumor suppressor proteins P53, pRB plays a major role in determining the fate of the infection

Cell cycle and tumor suppressors M=mitosis S=DNA synthesis phase G1=Gap between M and S G2=Gap between S and M

Taken from Principles of Virology Flint, Enquist, Krug, Racaniello and Skalka

Cell cycle and tumor suppressors M=mitosis S=DNA synthesis phase G1=Gap between M and S G2=Gap between S and M

P53,pRB

Cell cycle and tumor suppressors M=mitosis S=DNA synthesis phase G1=Gap between M and S G2=Gap between S and M

HPV E6 p53,pRB Ad E1A-B,E4

cancer

How does HPV cause cervical carcinoma • • •

E6 interacts with p53 preventing apoptosis and activating the cell cycle E7 interacts with pRB removing the brakes from the cell cycle In the spinous layers of the skin a rare integration event causes overexpression of E6 and E7 which can lead to abnormal tumor growth and ultimately cervical carcinoma (type 16,18, and 31’s E6 and E7 are apparently more oncogenic)

E6­Ap is a cellular protein found in association  With HPV E6

Finally, a high percentage of human tumors contain mutated p53 and / or pRB proteins. These mutations, which result from environmental Factors, reduce or abolish the ability of tumor suppressor Proteins to bind viral oncogenes and hence fail to Provide protection against transformation.

Cutaneous Warts • Common wart (verruca vulgaris): few mm in diameter, occur in large numbers anywhere in the skin, particularly hands, knees and feet. Usually do not cause inconvenience

• Flat warts (verrucae planae): predominantly children • Butchers’ warts: usually HPV-7, occupational hazzard

Genital warts

The most frequent in men and women is condylomata  Acuminata (“pointed lump”) In men: on the penis, urethra, anus and rectum (particularly In homosexuals). In women: on the vulva, vagina, cervix and anus. In children: external gangliata: sometimes indicators of  sexual abuse

penile

vulval

cervix

•  Cutaneous and genital warts are predominantly benign •  HPV DNA has been detected in cervical cell samples from     about 1/3 of normal young women and 10­fold lower     rates in women over 50 years. •  Epidemiologic  data support a central etiologic role of certain    sexually transmitted HPV types in cervical neoplasia. The risk     for genital HPV infections has been found associated with known    risk factors for cervical neoplasia: many sexual partners; early     age at first intercourse and use of oral contraceptives. •  Predisposition to malignancy is associated with some HPV types. •  Genetic factors are involved in 2 HPV­associated diseases affecting    the oral mucosa and skin: oral focal epithelial hyperplasia  (HPV    13 and 32) are highly prevalent in American Indians and Eskimos,    rarely in caucasians). EV (epidermodysplasia verruciformis) is    related to HPV 5 and 8.     

• HPVs are very stable • HPV infections include asymptomatic latent infections,      subclinical infections  and clinical manifestations. • Despite their amazing plurality, HPV subtypes are stable entities    with no evidence of inter/intra­typic recombination. • 68 HPV genotypes are recognized to date. By convention, a   “type” is distinct if less than 50% of its DNA is homologous   with other HPV type(s).  • “Types” are distinguished by cross hybridization or PCR. • Geographical distribution: world­wide. • They are highly host­specific: humans are not susceptible to    infection by animal papillomaviruses. • No cell culture system allows HPV propagation. Reason: they   grow in terminally­differentiating keratinocytes that cannot be   cultured.

Serological relationships between the 68 genotypes are little  Known. Reason: lesions contain very small amounts of virus and purified particles are hard to obtain.

Viral infections of mucosal Membranes • Eye Adenovirus (conjuctivitis) Sexually • Genital tract transmitted viruses HIV, HPV

HSV,

• Viruses live longer in damp environments • But must overcome mucosal immunity

Detection of Cervical Carcinoma • Papanicolaou smear (PAP smears) look for the presence of abnormally growing cells • If there are problems they may try to identify the presence and strain of papilloma virus (if it is high cancer type I.e. 16,18,31) further action may be taken • PCR and genotyping

The genomic organization of Ad5 and the E4 transcription unit.

Absence of E1A and E4 proteins and genes in the majority of cell lines transformed by E1A plus E4orf6 or E4orf3.

Transient coexpression of Ad5 E4orf6 or E4orf3 with Ad5 1A increases the mutation frequency at the hprt locus.