Viruses, Cells, and Diseases (CIOC5125Q)

Retrovirus (Raymond B. Birge, PhD) October 9th, 2008 Oncogenic Definition: A that is able to take residence in a 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 RNA Viral Genome DNA-dependent DNA polymerase (Viral) (Viral or Host) Viral mRNA Viral DNA Genome (integrated) DNA-dependent RNA polymerase RNA polymerase (Host) Viral Protein (Host) Viral RNA Genome RNA Splicing (Host) Examples: Viral Protein

Papovaviruses (Papillomavirus, SV40) Examples: Adenoviridae (Adenovirus) Oncovirinae (RSV, MuLV, HTLV-1,2) Herpesviridae (Epstein-Barr virus) Lentivirinae (HIV) TERMINOLOGY

Oncogene: A viral or cellular 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 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 ; Genome encodes three basic genes (Gag, Pol, and ) ALV (ASLV) Avian (Sarcoma) and Leukosis Virus

Coat proteins (surface glycoantigens); Encoded by Env Can be trans-membrane or cleaved. Env variations determine subgroups (A-E, and J). Outer lipid Envelope (Derived from the host membrane)

Polymerase (several genes; protease RT, RNase H, ) RNA genome (2 molecules) Capsid core proteins (core shell, includes the Matrix (MA) Capsid (CA-most abundant), and Nucleocapsid (NC)

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 Can be trans-membrane or cleaved. Env variations determine subgroups (A-E, and J). Outer lipid Envelope (Derived from the host membrane)

Polymerase (several genes; protease RT, RNase H, Integrase) RNA genome (2 molecules) Capsid core proteins (core shell, includes the Matrix (MA) Capsid (CA-most abundant), and Nucleocapsid (NC)

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)

Genus Type Hosts 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

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 ) is integrated into host 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 Retrovirus lifecycle is somewhat integrated DNA by Pol II. uneventful, consuming about 1% of the total cellular energy. 5. RNA is spliced and translated into protein

6. Virus particles assemble and bud from plasma membrane Retrovirus replication and integration:

5’ 5 ‘ 3’ 3’ Repeat Unique Single stranded RNA Unique Repeat

5 Cap (AAAA)n

Primer Polypurine binding Tract site 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 Retrovirus replication:

Primer Binding Site Polypurine Tract

5’ 5 ‘ 3’ 3’ Repeat Unique Single stranded RNA Unique Repeat Note, this sense strand does not serve directly as mRNA RT

tRNA primer binds to PBS; RT extends in 3’ direction

RT (only virus that uses tRNA for replication!)

RNase H degrades ds RNA (part of RT enzyme; degrades RT RNA/DNA duplex to ssDNA

SS DNA jumps to opposite RT complementary Strand

tRNA primer binds to PBS; RT RT extends in 3’ direction Retrovirus replication :

tRNA primer binds to PBS; RT RT extends in 3’ direction

Polypurine Tract

RT RNase H degrades all ds RNA except for the polypurine tract

RT RT now extends in the sense direction

RNA RT 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 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)

David Baltimore Howard Temin (MIT) (U Wisconsin)

1975 Nobel Prize in Physiology and Medicine The next step in virus 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

5’ LTR 3’ LTR

IN

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

Host DNA Viral DNA Host DNA host host LTR gag pol env LTR DNA provirus (through cellular transcriptional machinery) gag pol env 5’ 3’

gag-pol polyprotein gag polyprotein

Integrase; RT; Virion structural RHase H, protease proteins

Subgenomic env 5’ env 3’ mRNA env Envelope glycoproteins (SU and TM) A -causing virus in chicken

“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. RSV-Malignant sarcoma’s within 2 weeks ALV-Weakly transforming virus (tumors in 2-3 months) ALV and RSV could be propagated and isolated cultured cells

Normal cells RSV transformed cells

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 How can ALV, with a relatively uneventful lifecycle, induce cell transformation and neoplasia? The DNA Provirus Hypothesis (1965-74)

RNARSV DNARSV RNARSV Infecting virus Provirus Progeny Virus

The Oncogene Hypothesis (The protovirus hypothesis for origin of cancer genes )

DNA DNA RSV Altered DNA Provirus (~1971) Hidesaburo Hanafusa: Defectiveness of ; Virtually all acutely transforming of are mixtures of replication competent 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”. What is the difference between weak and acutely transforming viruses?

ALV RSV

Weakly transforming virus (3-6 months) Acutely transforming virus (1-2 weeks) Genome size ~8.5 kb Genome size ~10 kb Replication competent Replication deficient Origin of Retroviral Transforming Genes RSV (gag pol env src) AL V (gag,pol,env)

r.t. isolate genomic RNA

cDNA

src gag, pol etc gag, pol, env

DENATURE HYBRIDIZE

unhybridized hybridized sequences sequences src gag, pol, env

Stehelin, Bishop and Varmus Origin of Retroviral Transforming Genes

src Specific src probe

HYBRIDIZE

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

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

Stehelin, Bishop and Varmus Retrovirus and the Cancer Connection

J.Michael Biship Harold Varmus

1989 Nobel Prize in Physiology and Medicine Whats the difference between weak and acutely transforming viruses?

ALV RSV

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

Δenv gag pol env gag pol env src of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

LTR gag pol env LTR ALV DNA 8.5 kb

LTR gag pol src LTR RSV DNA env 10 kb

gag pol env src

gag & pol proteins Env proteins

Src proteins*

*Overexpression of mutated Src tyrosine kinase leads to cell transformation Avian Leukosis Virus Leukemogenesis by Promoter Insertional mutagenesis

transcription of myc from LTR ALV etc.

c-myc gene ALV Integration Site 5’ or 3’ of gene

ABERRANT REGULATION OF MMTV ‐int‐1 myc TRANSCRIPTION int‐2 Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)

LTR gag pol env LTR ALV DNA 8.5 kb

LTR gag pol src LTR RSV DNA env 10 kb

gag pol env src

gag & pol proteins Env proteins

Src proteins*

*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

Tyr527 c-Src Structure of v-Src vs c-Src genes

SH3 SH2 SH1 (PTK catalytic) v-Src

(open-high activity)

c-Src Tyr527 (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 (A-MuLV)

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

6 kB 9 kB

LTR LTR LTR LTR gag abl gag pol env

Mo-MuLV p120 Gag-Abl Helper or Dependovirus A-MuLV Tyrosine kinases are regulated by ‘auto-clamped’ structures using their SH2 and SH3 domains

c-abl c-Abl

Gag v-abl v-Abl

Bcr CML, ALL v-Src and v-Crk oncogene products

Crk was originally identified in the retroviral genome of avian retrovirus CT10

SH3 SH2 SH1 (PTK catalytic) p60src (RSV)

viral gag p47gag-crk (CT10)

PLC-γ II catalytic-1 catalytic-2 v-Crk ; CT10 regulator of kinase

.

1 208 248 340 373 424 440 p47gag-crk gag SH2 SH3

Phosphotyrosine Proline-rich sequence pYxxP Px(L)PxK,R

Binding partners* Binding partners* p130 Cas c-Abl CEF kDa CEF / v-Crk Paxillin C3G 200 c-Cbl SOS p130 Cas EGF-R DOCK180 116 AFAP Esp15 97 Gab1 JNK XWee1 Paxillin PI3 Kinase 66 * Partial List

45

31 Blot : α-P-Tyr Crk family of adaptor proteins

cAbl/Bcr-Abl

P P SH2 SH3-N Y SH3-C c-Crk II 222

SH2 SH3-N c-Crk I

Gag SH2 SH3-N v-Crk

P P SH2 SH3-N Y SH3-C Crk-L 207 The Field Comes Of Age

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.

Guanine-nucleotide exchange factor Guanine-nucleotide exchange factor

Ras-GDP Ras-GTP G12V Ras G12V Ras 98% 2% 0% 100%

GTPase activating protein (GAP) GTPase activating protein (GAP) Raf

ERK

AP-1 Activated Proto-oncogenes from DNA Transfection

Use Alu probe Compare sequence to NORMAL gene Isolate Human DNA

RESULT: RAS “Activation” RESULT: A SINGLE human gene is is due to a SINGLE point responsible for transforming mutation (gly val) at codon 12 capability

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

Parada and Weinberg Activated Proto-oncogenes from DNA Transfection

Human Bladder Tumor Isolate DNA (>99% mouse + cell line DNA 8-10 human genes)

Isolate high MW DNA restriction TRANSFORMATION endonuclease Isolate DNA TRANSFECTION fragments Isolate Human NIH 3T3 DNA fibroblasts TRANSFORMATION

Parada and Weinberg

Retrospective view of the Importance of Retroviral Oncogenes

• 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 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- oncogenes 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

EGF-R/HER2/Neu

RTK Inhibitors Ras Ras Inhibitors (MANY; ie Gefitinib) v-Ha-Ras v-Erb2 ABT-737 AG490 Src/PI3-kinase Raf v-p110 BAY439006 (Advanced kidney cancer) v-Src (Tyrosine kinase; Abl) v-Raf Gleevac-STI571 MAP-kinase

Nu2048 (Myc) Transcription of growth gene v-Myc Memorial symposium for the contributions of Teruko Hanafusa (1998)

Nature Reviews Molecular Cell Biology 2; 467-475 (2001); THE HUNTING OF THE SRC Homework Assignment: Due October 16th

Now that you are experienced virologists (after passing “Viruses, Cells, and Diseases”!),you have been invited to work on novel oncogenic 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.

1. How would you determine whether the virus is a retrovirus? What assays would you do, and how would you interpret your data?

2. 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! Milestones in Cancer Biology.

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-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 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)