Retroviruses May 14 th 2015

Retroviruses May 14 th 2015 I. Retroviruses Overview Retroviruses are termed retro because they encode a reverse transcriptase enzyme. It s apt, because they start with an RNA genome, which is reverse transcribed into DNA i.e. reverse of the natural flow of DNA to RNA to proteins. Retroviruses are widespread and found in humans, mammals, birds, fish, worms etc. They cause: Tumors e.g. HTLV, Rous sarcoma virus (RSV), murine leukemia virus (MLV). Immunodeficiencies e.g. HIV, other lentiviruses, simian retroviruses (SRVs). Neurological disease including dementias e.g. HIV, other lentiviruses, HTLV Arthritis e.g. lentiviruses including Caprine arthritis encephalopathy virus (CAEV). 1) Classification Early classification was based on structure and how they budded from infected cells in electron micrographs e.g. B-type, C-type etc. This was replaced by a system that reflects their relationships based on sequence. Family Retroviruses Genus Example Group Alpha retroviruses Avian leucosis virus ALV Simple Beta retroviruses Murine mammary tumor virus MMTV Simian retroviruses SRV Gamma retroviruses Murine leukemia virus MLV Delta retroviruses Human T-cell leukemia virus HTLV Complex Epsilon retroviruses Walleye dermal sarcoma virus Lentiviruses Human immunodeficiency virus HIV Spumaviruses Simian foamy virus SFV 1

2) Retroviral genes Simple retroviruses Basic simple gene structure. gag-pol-env MLV (group antigen Gag, polymerase and other enzymes, envelope) Complex retroviruses Have additional genes termed regulatory (tat and rev for HIV-1) or accessory (vif, vpr, vpu and nef) gag-pol-env-tat-rev-nef-vif-vpu-vpr gag-pol-env-tat-rev-nef-vif-vpr-vpx gag-pol-env-tax-rex-hbz HIV-1 HIV-2/SIV HTLV 3) Virion Structure Components Envelope: Enveloped, lipid membrane, envelope spikes consist of surface SU and transmembrane TM proteins and arranged as trimers. Gag: Myristylated matrix MA protein underneath the membrane. In HIV, the envelope has been shown to interact with MA. Envelope spikes may fit into depressions on oligomeric MA. Capsid CA, NC nucleocapsid Many NC molecules cover the two molecules of RNA per virion. Enzymes PR protease RT reverse transcriptase IN integrase RNA two copies. RNA: Cap-R-U5----------PBS--DLS--Ψ----GAG-POL------ENV------U3-R-AAAAAAAA Dimer linkage structure DLS Primer binding site PBS Ψ is packaging signal U5 and U3 are unique regions at either end of genome R is repeat region. 4) Retrovirus replication cycle ---attachment, entry and uncoating Virus must bind to cell, and needs to induce fusion of viral membrane and cell membrane. 2

For mammalian retroviruses, fusion is predominantly caused by interactions between the Env spikes and cell surface receptors. Most mammalian retroviruses including HIV and HTLV are ph independent. Some uncoating events then occur allowing the viral core to move into the cytoplasm. Unclear what is happening here. However, a structure called the reverse transcriptase complex (RTC) begins to traffic via the cytoskeleton towards the nucleus. --- Reverse transcription and integration Reverse Transcriptase: RT is encoded by the pol gene, which also encodes integrase and protease. Structure/ Functions RT also has RNAse H activity. heterodimer p51/p66, identical except p51 lacks the RNAse H activity. Virus particles have two molecules of +ve stranded RNA but also carry host transfer RNAs that act to prime the synthesis of DNA strand. RT catalyzes the formation of a double stranded DNA molecule with long terminal repeats LTRs, where the U5, R and U3 regions are repeated at each end of the cdna molecule. The structure carrying the cdna is called the pre-integration complex (PIC). ---Integration Double stranded linear forms carrying 2 x LTRs are integrated into chromosomal DNA. Integration is done by the integrase. Integration is frequently described as non-specific. However, there is some specificity. MLV integrates near transcription start sites and CpG islands (in promoters). HIV usually integrates along transcriptionally active genes. This difference is due to host proteins exploited by the PIC to dock onto chromosomes. The HIV PIC binds LEDGF. --- RNA transcription Retroviruses exploit the cellular RNA polymerase, RNA pol II for mrna synthesis. Transcription runs from LTR to LTR: LTR LTR 3

Two mrnas are produced in most simple retroviruses Gag/Pol mrna Env mrna Usually a single promoter in the LTR. U3 R U5 AP-1 NF-AT NF-kB SP-1 TATA TAR AAUAAA NRF (IL2/NRE) HIV-1 LTR region/promoter transcription For simple retroviruses, one mrna is a full length genome and the other encodes just Env. The retroviral mrnas are capped and poly adenylated. The full length mrna acts either as a mrna or is packaged as the viral genome into virus particles. The env mrna acts only as a messenger RNA and is not packaged. The splice donor is upstream from the Ψ packaging signal, so that env mrna doesn t have a packaging signal. --- Translation, proteins and assembly Two proteins are translated from the full length mrna; these are Gag and Gag/Pol precursor proteins The precursor Gag protein contains matrix MA, capsid, CA and nucleocapsid NC. The precursor Gag/Pol contains MA, CA, NC as well as protease, RT and integrase. The precursors are not cleaved into constituent proteins until the virion has budded. MLV exploits translational readthrough that allows the ribosome to translate beyond a stop codon at the end of Gag to translate Gag/Pol. HIV uses a frame shift mechanism so that the ribosome slips (-1 frame) into a second reading frame and translates Gag/ Pol as an extra long protein. A sequence that includes several Us (UUUUUUA) is the frameshift site for HIV. These mechanisms mean that about 20x as much Gag is made as Gag/Pol. 4

Env is made as a type I membrane protein with a leader sequence and a single TM region. It is secreted and assembled as trimers in the ER and cleaved into TM and SU. For HIV, host furin proteases (subtilisin-like serine protease) in the Golgi do this, recognizing a K/RXK/RR cleavage site. ---Packaging of viral RNA Ψ packaging signals Ψ signals are recognized by zinc fingers in NC; 2 for HIV NC 1 for MLV NC Once one NC is on; then the other NCs oligomerize, lining up along the RNA. Remember, NC described here is still part of the Gag precursor. ---Budding viruses Retroviruses bud from cell membranes. Budding requires host cell proteins and is regulated by so-called late domains. Late domains are short amino acid motifs with the following sequences: PT/SAP PPXY YPDL HIV-1 p6 is an additional Gag protein that carries two late domain sequences. Late domains recruit cellular ESCRT (endosomal sorting complexes required for transport) proteins that are required for budding e.g. HIV-1 p6 recruits tsg101. Mutation of late domains leads to accumulation of incompletely budded particles at the cell surface. ---Virion maturation Budded virions are not infectious without maturation. This is the stage that the viral protease cleaves the Gag and Gag/Pol precursors into their constituent proteins. Protease inhibitors widely used for HIV treatment, block at this stage. 5) Transformation ---Transformation by acute transforming viruses These are retroviruses that have incorporated (i.e. transduced) host genes into their genomes. These host genes or oncogenes become mutated and their expression out of context leads to transformation. Examples include Rous sarcoma virus, Harvey murine sarcoma virus RSV has incorporated src. Harvey MSV has incorporated p21ras There are examples of various retroviruses that have incorporated various host genes involved in control of cell division. (See chapter in Field s Virology (2007 or 2013). The 2007 edition is available online via Lamar Soutter Library Website. 5

Examples include: growth factor receptors, signaling kinases, transcription factors All are involved in regulating cell division. Transduction of cellular genes in this way is a very rare event. Rous sarcoma virus carries a src gene incorporated into an infectious genome. However, for most transforming retroviruses, oncogenes are incorporated into genomes that have lost some or all viral genes and are replication incompetent alone. Such genomes carry an oncogene between 2 LTRs and will also carry a packaging signal. LTR Ψ oncogene LTR They can only replicate in the presence of a full-length replication competent virus that will express all the proteins required to make a virus particle. Replication incompetent viruses that require a helper virus are not transmitted very efficiently. Acutely transforming retroviruses have often been identified in animals bred at high density e.g. chickens. Not yet observed in humans (who also live in dense populations). ---Transformation and tumors caused by insertional mutagenesis These tumors are caused by retroviruses that do not carry an oncogene. Leukemias caused by ALVs, MLVs, breast tumors by MMTV. Develop slowly, are caused by integration events in inappropriate places. Probably is one step in a multistep process Thus the integration disrupts or activates a gene that contributes to leukemia, cancer. Several ways this can happen: 1. Promoter insertion. Inserts a promoter upstream from a gene or inside a gene. e.g. gene with 4 exons: The provirus inserts in the middle. In the example shown below, transcription from 3 LTR will yield an mrna with the two 3 exons and will result in a novel protein being produced. LTR LTR 2. Enhancer insertion. The LTRs carry enhancer regions, which can activate genes when inserted in the vicinity. 6. Retroviral vectors and gene therapy Retroviral vectors aim to deliver a retrovirus-based genome that will integrate into the host cell chromosome 6

and express only the gene of choice. Widely used in research and have applications in gene therapy. Historically, this was achieved by constructing "so-called" packaging cell lines. Packaging cell gag-pol gag-pol 7

Delivery of a therapeutic gene or gene of choice RNA DNA Vectors based on MLV can only introduce therapeutic genes into dividing cells. Retroviral vectors based on HIV and other lentiviruses that can infect non-dividing differentiated cells e.g. macrophages, have also being developed. Envelope glycoproteins There is a choice of different Envs. For gene therapy of humans, an Env that infects human cells is needed. The amphotropic MLV Env infects human cells and is frequently used for vectors and gene therapy approaches. Other possibilities include VSV-G that confers infection of a wide range of cell types. ---X-linked severe combined immunodeficiency Correction by gene therapy and resulting leukemias 1. X-linked severe immunodeficiency (XL-SCID) results from a lesion in the gene for the common γc chain that is a subunit for several interleukin receptors e.g. IL-2, Il-4, IL-7. A mature immune system is not formed. Children with this defect must live in isolation tents. 2. 19 XL-SCID children were treated in two trials in Paris and London. An MLV based retroviral vector carrying γc was used. 8

3. Bone marrow stem cells were removed and infected. 4. Infected cells were re-infused so that they would repopulate and hopefully restore immune competency. For 17 of the 20 children, the treatment was successful and they were able to resume normal lives outside isolation. Several years after treatment, five children have developed T-cell leukemia; one died. The leukemic cells originated from the re-infused T-cells. In four of the leukemias, the provirus was close to a cellular gene called LMO2. The role of LMO2 1. LMO2 protein acts as a bridging or scaffold molecule in transcription factor complexes. 2. LMO2 acts early in hematopoiesis. Mice without LMO2 fail to develop hematopoietic cells of any lineage. 3. Deregulation of LMO2 expression has been associated with human T-cell acute leukemias following chromosomal translocations. 4. Deregulation of LMO2 expression has also been associated with leukemias in mice caused by MLV retrovirals insertions. Safer Vectors A new trial for treatment of XL-SCID has begun. The new trial uses a vector, which does not contain an enhancer region in the LTR. Eventually vectors that mediate precise editing of host gene mutations will likely be the therapy of choice, 9

II Human T-cell leukemia viruses HTLV-I discovered in 1981 by US and Japanese groups. HTLV-II HTLV-3 (very few) HTLV-4 (1 case) STLVs Complex Retroviruses. HBZ Regulatory genes Tax transactivates HTLV LTRs. How does tax work? Tax acts on the HTLV LTR and stimulates transcription. Tax binds GC rich regions on the LTR upstream from CRE sites, interacting with adjacent CREB transcription factors. However, Tax has effects on several transcription factors including NF-Kβ, where it stimulates its dissociation with IKβ. Rex In mammals, mrnas are fully spliced unless splicing is controlled. Early in infection, HTLV produces multiply spliced mrnas that encode Tax and Rex. When sufficient Rex is produced, it binds a secondary RNA structure (the rex response element, RxRE) on HTLV mrnas and direct 10

unspliced and singly spliced mrnas away from the spliceosome and out of the nucleus. These latter mrnas encode the structural proteins of the virus particle. HTLV Rex is functionally equivalent to HIV s Tat. 1) HTLV-I Prevalence and disease About 20 million people worldwide are estimated to be infected. HTLV-I is particularly prevalent in Japan, the Caribbean and Central Africa. In some areas of Japan, over 30% of the population are seropositive. HTLV is also present in IV drug abusers. Less than 3% of infected individuals will suffer an HTLV-1 related disease in their lifetime. Adult T-cell leukemia About 1% of HTLV-I+ individuals eventually suffer adult T-cell leukemia (ATL). Tumors are usually derived from CD4+ T-cells. Tumor cells carry an integrated HTLV-I provirus. Individual tumors are usually clonal, but sites of integration vary between tumors. Thus, the mechanism of tumor formation is NOT insertional mutagenesis. Tax is intimately involved in tumorigenesis and upregulates IL-2, IL-2Rα, c-myc, c-sis and other genes involved in growth regulation. Genes repressed include p18ink4 and p53, proteins also involved in cell cycle regulation. Tax alone can transform T-cells in vitro. However, tax is not expressed in all ATL tumors. A newly discovered HTLV-I gene, HBZ, is transcribed from the negative strand of the provirus. HBZ induces cell proliferation like tax and is expressed in 100% of tumors. ATL takes years to develop and does not develop in the vast majority of HTLV-I+ individuals. Thus further additional changes must be needed for a malignant T-cell leukemia to develop. HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Neurological disease; weakness, spasticity, incontinence, sensory loss and demyelination. Up to 1% HTLV+ individuals develop HAM Little infection of CNS cells. HAM is associated with more intense humoral and cellular immune responses. In HAM, the frequency of HTLV-specific CD8 T-cells in CSF can exceed that in the blood. High titers of HTLV specific antibodies are detected in CSF. Inflammatory or autoimmune mechanisms have been suggested. HTLV-II HTLV-II is a related but genetically distinct virus. Much rarer than HTLV-I. HTLV-II is prevalent at low frequency in Amerindian populations of America and (as for HTLV-I) is found in intravenous drug abusers. HTLV-II was first discovered in an atypical hairy cell leukemic cell line and has been associated with an extremely small number of other hairy cell leukemias. HTLV-II Is more frequently associated with HAM than hairy cell leukemias. 11

Further Reading and questions These notes cover what you need to know. Field s Virology (2007) is available online where you can search if you have questions. A new 2013 version of this textbook is also available. Feel free to contact me if you have questions: (paul.clapham@umassmed.edu). 12