Viruses, Cells, and Diseases (CIOC5125Q) Retrovirus (Raymond B. - - PowerPoint PPT Presentation

Retrovirus (Raymond B. Birge, PhD) October 9th, 2008

Viruses, Cells, and Diseases (CIOC5125Q)

Oncogenic Viruses

Definition: A virus that is able to take residence in a cell and alter cellular growth and give properties of neoplasia. These cells are referred to as “transformed” cells

DNA Tumor Viruses RNA Tumor Viruses

DNA Viral Genome Viral mRNA Viral Protein

DNA-dependent DNA polymerase (Viral or Host) RNA polymerase (Host)

Examples: Papovaviruses (Papillomavirus, SV40) Adenoviridae (Adenovirus) Herpesviridae (Epstein-Barr virus) RNA Viral Genome Viral DNA Genome (integrated) Viral RNA Genome Viral Protein

Reverse transcriptase (Viral) DNA-dependent RNA polymerase (Host) RNA Splicing (Host)

Examples: Oncovirinae (RSV, MuLV, HTLV-1,2) Lentivirinae (HIV)

TERMINOLOGY

Oncogene: A viral or cellular gene directly responsible for the induction of abnormal cell proliferation. Proto-Oncogene: A cellular gene that has the potential to be an oncogene if its encounters mutation/de-regulation. Tumor Suppressor Gene: A cellular gene involve in growth suppression. These genes are inactivated by deletion, mutation, of interaction with viral proteins (for example, HPV gene products). Oncogene Hypothesis: Malignant carcinoma’s arise from de-regulation or mutation of cellular genes.

Basic Structure of Retrovirus; Genome encodes three basic genes (Gag, Pol, and Env) ALV (ASLV) Avian (Sarcoma) and Leukosis Virus

Coat proteins (surface glycoantigens); Encoded by Env Outer lipid Envelope (Derived from the host membrane) Can be trans-membrane or cleaved. Env variations determine subgroups (A-E, and J). Capsid core proteins (core shell, includes the Matrix (MA) Capsid (CA-most abundant), and Nucleocapsid (NC) RNA genome (2 molecules) Polymerase (several genes; protease RT, RNase H, Integrase)

RNA genome; both RNA molecules are single stranded (+) sense and 5’ cap and 3’ poly A tail. They also have a small molecule of tRNA (usually for Trp or Lys!)

Basic Structure of Retrovirus; Genome encodes three basic genes (Gag, Pol, and Env) ALV (ASLV) Avian (Sarcoma) and Leukosis Virus

Coat proteins (surface glycoantigens); Encoded by Env Outer lipid Envelope (Derived from the host membrane) Can be trans-membrane or cleaved. Env variations determine subgroups (A-E, and J). Capsid core proteins (core shell, includes the Matrix (MA) Capsid (CA-most abundant), and Nucleocapsid (NC) RNA genome (2 molecules) Polymerase (several genes; protease RT, RNase H, Integrase)

Coat proteins also determine trophism

Coat proteins determine trophism; Ecotrophic=infects mouse Xenotrophic=infects non mouse (rat, hamster) Amphotrophic=mouse and non-mouse (human)

Topology and classification of retrovirus

A-type: Non-enveloped particles, only seen inside cells (maybe they are partially expressed endogenous virus) B-type: Enveloped particles, with condensed core and prominent envelope spikes (MMTV) C-type: Enveloped particles, with condensed core and few envelope spikes (ALV, RSV, HIV, HTLV) D-type: Enveloped particles, less condensed core, few envelope spikes (able to super-infect C-type virus)

Taxonomy of RNA Reverse Transcribing Viruses (Family=Retroviridae)

Alpharetrovirus ALV, RSV, CT10, Y73 Sarcoma Virus Vertebrate Betaretrovirus MMTV, Squirrel monkey retrovirus Vertebrate Gammaretrovirus FLV, Harvey MSV, Moloney MSV Vertebrates Deltaretrovirus BLV, Primate T-lymphocytic retrovirus Vertebrate Lentivirus HIV-1, HIV-2, SIV, HTLV Vertebrates Spumavirus Chimp foamy virus Vertebrates

Genus Type Hosts

Classification taxonomy depend on various factors that include genome size, assembly mechanisms, subtype, malignancies, immunodeficiency, and homology.

The basic retrovirus lifecycle

The retrovirus lifecycle in more detail

• 1. Binding to a receptor (fusion/internalization)

Note, there are four subtypes (A-type, B-type, C-type, D-type)

• 2. RNA (plus strand) is copied to DNA

(minus)strand. ssDNA copies to dsDNA

• 3. DNA, called provirus) is integrated into

host chromosome randomly

• 4. Full-length genomic RNA is copied from

integrated DNA by pol II.

• 5. RNA is spliced and translated into protein
• 6. Virus particles assemble and bud from

plasma membrane

The lifecycle in more detail

• 1. Binding to a receptor (fusion/internalization)

Note, there are several subtypes (A-type, B-type, C-type, D-type etc)

• 2. RNA (plus strand) is copied to DNA

(minus)strand. ssDNA copies to dsDNA

• 3. DNA, called provirus) is integrated into

host chromosome randomly

• 4. Full-length genomic RNA is copied from

integrated DNA by Pol II.

• 5. RNA is spliced and translated into protein
• 6. Virus particles assemble and bud from

plasma membrane

Retrovirus lifecycle is somewhat uneventful, consuming about 1%

• f the total cellular energy.

Retrovirus replication and integration:

5’ Repeat 3’ Repeat 5 ‘ Unique 3’ Unique Primer binding site Polypurine Tract Single stranded RNA

Major elements of the retrovirus sense strand. Direct repeats at both ends of the genome ‘terminally redundant” Primer binding site, uses a specific tRNA (15-20 nucleotides C’ to 3’ end) Polypurine Tract; Short stretch of A and G residues for initiating (+) strand synthesis Unique 3’ region responsible which forms the promoter for the viral mRNA

5 Cap (AAAA)n

RT

Retrovirus replication:

5’ Repeat 3’ Repeat 5 ‘ Unique 3’ Unique Primer Binding Site Single stranded RNA RT RT RT RT

tRNA primer binds to PBS; RT extends in 3’ direction (only virus that uses tRNA for replication!) RNase H degrades ds RNA (part of RT enzyme; degrades RNA/DNA duplex to ssDNA SS DNA jumps to opposite complementary Strand tRNA primer binds to PBS; RT extends in 3’ direction

Polypurine Tract

Note, this sense strand does not serve directly as mRNA

Retrovirus replication :

RT

tRNA primer binds to PBS; RT extends in 3’ direction RNase H degrades all ds RNA except for the polypurine tract

RT Polypurine Tract

RT now extends in the sense direction

RT RT

RNA RNase H removes remaining RNA C’ sequences allow for circularization

After circularization, RT acts as a DNA-dependent DNA polymerization to make the ds DNA pro-virus

David Baltimore (MIT) Howard Temin (U Wisconsin)

RSV virus particles contain an endogenous DNA polymerase activity

(incorporates deoxyribonucleside monophosphates into DNA and requires all four deoxyribonucleotide triphosphates, a divalent cation,and is inactivated by RNAase)

1975 Nobel Prize in Physiology and Medicine

The next step in virus life is the DNA provirus integration, catalyzed by the integrase function of RT. Integrase Function is part of RT, proviral insertion Can occur with either linear of circular form of the provirus

The ends of the LTR’s have inverted repeats that are cleaved to form a staggered cut. 5’ LTR 3’ LTR IN IN also makes a cut in the host cell DNA, allowing permanent insertion

• f the entire ds provirus

Host DNA Host DNA Viral DNA

DNA provirus host host

LTR LTR

gag pol env

5’ 3’

gag pol env

Transcription (through cellular transcriptional machinery)

gag-pol polyprotein gag polyprotein

Virion structural proteins Integrase; RT; RHase H, protease Envelope glycoproteins (SU and TM)

env

5’ 3’

env

Subgenomic env mRNA

“A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to wide-spread metastasis. In a careful study of the growth, tests have been made to determine whether it can transmitted by a filtrate free of the tumor cells… small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl” (Rous, Nature , 1911). The cancer was named Rous' sarcoma, and Rous won the Nobel Prize in 1966 for his achievement.

A cancer-causing virus in chicken

RSV-Malignant sarcoma’s within 2 weeks

ALV-Weakly transforming virus (tumors in 2-3 months)

Growth characteristic Normal cells Tumor cells Density dependent inhibition of growth Present Absent Growth factor requirement High Low Anchorage dependence Present Absent Proliferative life span Finite Indefinite Contact inhibition of motility Present Absent Morphology Flat Rounded Colonies in agar NO YES Normal cells RSV transformed cells

ALV and RSV could be propagated and isolated cultured cells

How can ALV, with a relatively uneventful infection lifecycle, induce cell transformation and neoplasia?

The DNA Provirus Hypothesis (1965-74) RNARSV DNARSV Infecting virus RNARSV Provirus Progeny Virus The Oncogene Hypothesis (The protovirus hypothesis for

• rigin of cancer genes)

DNA Altered DNA DNARSV Provirus

Hidesaburo Hanafusa: Defectiveness of Rous sarcoma virus; Virtually all acutely transforming retroviruses of animals are mixtures of replication competent helper virus and replication defective transforming virus.Replication function is provided by the helper virus in trans

Hybridization experiments. PNAS (1970). “These results indicate that both cell types (chicken cells that contain RAV-60 in a replicating form or do not appear to contain a replicating form) contain DNA that is complementary to RNA from the avian tumor virus”.

(~1971)

ALV RSV

Weakly transforming virus (3-6 months) Genome size ~8.5 kb Replication competent Acutely transforming virus (1-2 weeks) Genome size ~10 kb Replication deficient

What is the difference between weak and acutely transforming viruses?

Origin of Retroviral Transforming Genes

RSV (gag pol env src)

r.t.

gag, pol etc

src

cDNA

DENATURE

AL V (gag,pol,env)

isolate genomic RNA

gag, pol, env HYBRIDIZE

unhybridized sequences hybridized sequences

gag, pol, env src

Stehelin, Bishop and Varmus

src

Origin of Retroviral Transforming Genes

Specific src probe HYBRIDIZE RSV-Infected CEF (+ control) “Normal” chick DNA MOUSE DROSOPHILA HUMAN

+ + + + +

Stehelin, Bishop and Varmus

Thus: a proto-oncogene is the NORMAL progenitor gene of a viral oncogene

J.Michael Biship Harold Varmus 1989 Nobel Prize in Physiology and Medicine

Retrovirus and the Cancer Connection

ALV RSV

Weakly transforming virus (3-6 months) Genome size ~8.5 kb Replication competent Acutely transforming virus (1-2 weeks) Genome size ~10 kb Replication deficient

Whats the difference between weak and acutely transforming viruses? gag env pol Δenv gag pol env src

LTR LTR

gag pol env

LTR LTR

gag pol env src

8.5 kb 10 kb

ALV DNA RSV DNA

pol env src gag

gag & pol proteins Env proteins Src proteins*

Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

*Overexpression of mutated Src tyrosine kinase leads to cell transformation

Avian Leukosis Virus Leukemogenesis by Promoter Insertional mutagenesis

ALV

ALV Integration Site 5’ or 3’ of gene c-myc gene transcription of myc from LTR ABERRANT REGULATION OF myc TRANSCRIPTION MMTV ‐int‐1 int‐2 etc.

LTR LTR

gag pol env

LTR LTR

gag pol env src

8.5 kb 10 kb

ALV DNA RSV DNA

pol env src gag

gag & pol proteins Env proteins Src proteins*

Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

*Overexpression of mutated Src tyrosine kinase leads to cell transformation

Src and tyrosine phosphorylation (Brugge and Hunter)

RSV encodes a 60 kD protein called Src Injected RSV-bearing tumors into rabbits--generated anti-sera against 60 kD protein Using a technique called Immunoprecipitation, investigators showed that this anti-sera bound a protein with an unusual kinase specificity-- Shortly thereafter, many oncogenes identified in RNA viruses encoded tyrosine kinases.

In cells transformed by many Src and Abl (and other TK oncogenes) There is dramatic increase in protein tyrosine phosphosprylations

Structure of v-Src gene

SH3 SH2 SH1 (PTK catalytic) p60src (RSV)

Structure of v-Src vs c-Src genes

SH3 SH2 SH1 (PTK catalytic) v-Src c-Src

Tyr527

Structure of v-Src vs c-Src genes

SH3 SH2 SH1 (PTK catalytic) v-Src c-Src

Tyr527

(open-high activity) (closed-suppressed activity)

Non-receptor tyrosine kinases are generally maintained in auto-inhibited clamped structures, but very sensitive to mutational activation and global destabilization.

Kuriyan, Rockefeller University, UC Berkely

Abelson leukemia virus encodes a cellular oncogene called abl, another non-receptor tyrosine kinase (like Src) Abelson murine leukemia virus (A-MuLV) Derived from Moloney murine leukemia virus (Mo-MuLV) Genus= gamma-retrovirus Causes a rapidly progressive lymphosarcoma in mice

6 kB

LTR LTR

gag abl 9 kB

LTR LTR

gag p120 Gag-Abl pol env A-MuLV Mo-MuLV Helper or Dependovirus

Tyrosine kinases are regulated by ‘auto-clamped’ structures using their SH2 and SH3 domains

Gag Bcr CML, ALL

c-abl c-Abl v-abl v-Abl

v-Src and v-Crk oncogene products

SH3 SH2 SH1 (PTK catalytic) viral gag p60src (RSV) p47gag-crk (CT10) PLC-γ II catalytic-1 catalytic-2 Crk was originally identified in the retroviral genome of avian retrovirus CT10

SH2 gag SH3

1 440 208 248 340 373 424

Phosphotyrosine pYxxP Proline-rich sequence Px(L)PxK,R p130 Cas Paxillin c-Cbl EGF-R Gab1 XWee1 c-Abl C3G SOS DOCK180 Esp15 JNK PI3 Kinase

v-Crk ; CT10 regulator of kinase

p47gag-crk

97 66 45 kDa

116

CEF / v-Crk

Blot : α- P-Tyr

CEF

31 200

p130 Cas Paxillin

Binding partners* Binding partners* * Partial List

AFAP

cAbl/Bcr-Abl SH2 SH3-C SH3-N Y P P

222

c-Crk II SH2 SH3-N c-Crk I SH2 SH3-N Gag v-Crk SH2 SH3-C SH3-N Y P P Crk-L

207

Crk family of adaptor proteins

Harvey and Kirsten murine sarcoma viruses

Originally discovered in 1960’s by Jennifer Harvey and Weiner Kirsten (Harvey murine sarcoma virus and the Kirsten sarcoma virus) Both viruses encoded retroviral genomes with the h-ras or k-ras cellular genes. The protein products were 21 kD GTPases and later shown to have G12V mutations.

Ras-GDP Ras-GTP

Guanine-nucleotide exchange factor GTPase activating protein (GAP) Guanine-nucleotide exchange factor GTPase activating protein (GAP)

G12V Ras G12V Ras 98% 2% 100% 0%

The Field Comes Of Age

Raf ERK AP-1

Activated Proto-oncogenes from DNA Transfection

RESULT: RAS “Activation” is due to a SINGLE point mutation (gly val) at codon 12

Use Alu probe

Isolate Human DNA

RESULT: A SINGLE human gene is responsible for transforming capability

Sequence

RESULT: The gene is the HUMAN c-H-ras gene !!!

Compare sequence to NORMAL gene

Parada and Weinberg

Activated Proto-oncogenes from DNA Transfection

Human Bladder Tumor cell line DNA Isolate high MW DNA Isolate DNA fragments

restriction endonuclease

TRANSFECTION

NIH 3T3 fibroblasts

TRANSFORMATION

Isolate DNA (>99% mouse + 8-10 human genes)

TRANSFORMATION

Isolate Human DNA

Parada and Weinberg

• 1984- Avian erythroblastosis virus gene

(v-erbB) is shown to be a truncated Epidermal Growth Factor receptor

• 1985- Simian sarcoma virus transforming gene (v-sis) is

shown to be analogous to the β chain of PDGF receptor

• 1987- v-jun Avian oncogene is shown to be analogous to

AP-1 Many of the Proto-oncogenes in viruses turned out to be involved in sporadic mutations in human cancers

Retrospective view of the Importance of Retroviral Oncogenes

About 25 naturally occurring oncogenes have been identified

Oncogenes in the 21st Century

• STI 571 (Gleevec, Imatininb) is the first line therapy

for CML

– The target protein is Bcr-Abl, a protein tyrosine kinase (Abl was first known from a retrovirus) – Therapy resistance stems from changes in other proto-

• ncogenes such as Src

– Second line and combination therapies with Src inhibitors are becoming standard

• BUT-there are failures as well as successes-EGF-R

inhibitors are of limited value, so there is still much to learn

The EGF-R-Ras-Raf-Map kinase pathway is an important targeting module for cancer therapeutics

BAY439006 (Advanced kidney cancer)

Raf MAP-kinase Transcription of growth gene Src/PI3-kinase (Tyrosine kinase; Abl) EGF-R/HER2/Neu Ras Ras Inhibitors RTK Inhibitors (MANY; ie Gefitinib) Gleevac-STI571 AG490 Nu2048 (Myc)

v-Erb2 v-Ha-Ras v-Raf v-Myc v-Src

ABT-737

v-p110

Memorial symposium for the contributions of Teruko Hanafusa (1998)

Nature Reviews Molecular Cell Biology 2 ; 467-475 (2001); THE HUNTI NG OF THE SRC

Homework Assignment: Due October 16th

1. How would you determine whether the virus is a retrovirus? What assays would you do, and how would you interpret your data? Now that you are experienced virologists (after passing “Viruses, Cells, and Diseases”!),you have been invited to work on novel

• ncogenic virus (called C5125Q) isolated from a mouse population

in the Outback of Australia. Injection of serum or purified virus causes a acute leukemia, specific to T and B cells. Using both specific knowledge (and your imagination) describe a scenario by which C5125Q may cause transformation. Remember, this is a novel retrovirus and not likely to behave exactly as the viruses we studied in class! 2.

• 1838. Recognized as a collection of disorganized, abnormal cells (Virchow)

1907-33. Cell-free filtrates induce tumors in chickens (Rous, Shope, Fujimani) 1910-25 Chromosomal abnormalities (Tyzzer, Boveri). 1915-41 Carcinogen-induced cancers, Multiple factors involved--Initiation and Promotion factors (Yamigiwa, Berenblum) 1951-53 Mouse Leukemia Virus, Polyoma Virus (Gross, Friend, Stewart)

• 1964. Provirus Hypothesis (Temin)

1969-76. Normal cells suppress transformation--cell hybrids (Harris,Stanbridge)

• 1970. Reverse Transcriptase (Baltimore, Temin)
• 1971. Hereditary nature of retinoblastoma (Knudsen)

1976-80. Oncogene theory, cellular origin of viral transforming genes (Varmus, Bishop, Hanafusa, Vogt).

Milestones in Cancer Biology.

Milestones in Cancer Biology-part II 1979-1982. Discovery of Ras oncogene (Weinberg, Cooper, Wigler, Barbacid) 1982-1987. Activation of many genes by virus integration, many oncogenes identified.

• 1988. Isolation of Retinoblastoma gene--concept of ‘tumor suppressor genes’
• developed. (Weinberg)

1988-1991. Multistep carcinogenesis models (Vogelstein, Liotta) 1992-1994. Programmed cell death, Bcl-2, p53 (Korsmeyer, Levine, Horvitz)

• 1996. Angiogenesis Inhibitors (Folkman)
• 1999. STI-571 (Glivac) Bcr-Abl Inhibitor, CML (Drucker, Kuriyan)

2001-06. Era of Genomics, Proteomics, to the cancer problem (Collins, Ventor, Lander)

• 2007. Cancer stem cell biology and tumor microenvironment (Bissell/Weinberg)