Viruses, Cells, and Diseases (CIOC5125Q) Retrovirus (Raymond B. Birge, PhD) October 9th, 2008
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 RNA Viral Genome Reverse transcriptase DNA-dependent DNA polymerase (Viral) (Viral or Host) Viral mRNA Viral DNA Genome (integrated) DNA-dependent RNA polymerase RNA polymerase (Host) (Host) Viral Protein 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 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 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) 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 ) Type Hosts Genus 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 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 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: 3’ 5’ 3’ 5 ‘ Single stranded RNA Unique Repeat Repeat Unique 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 3’ 5’ 3’ 5 ‘ Single stranded RNA Unique Repeat Repeat Unique Note, this sense strand does not serve directly as mRNA RT tRNA primer binds to PBS; RT extends in 3’ direction (only virus that uses tRNA for RT 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 RNase H degrades all ds RNA RT 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 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 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 Viral DNA Host DNA Host DNA
host host LTR LTR gag pol env DNA provirus Transcription (through cellular transcriptional machinery) gag pol env 5’ 3’ gag-pol polyprotein gag polyprotein Integrase; RT; Virion structural RHase H, protease proteins env Subgenomic env 3’ 5’ mRNA Envelope glycoproteins env (SU and TM)
A cancer-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 infection lifecycle, induce cell transformation and neoplasia?
The DNA Provirus Hypothesis (1965-74) RNA RSV DNA RSV RNA RSV Infecting virus Provirus Progeny Virus The Oncogene Hypothesis (The protovirus hypothesis for origin of cancer genes�) DNA RSV Altered DNA DNA Provirus
(~1971) 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”.
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