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.