11-16-09 Introduction to Retroviruses Basic characteristics of a retrovirus: Enveloped Diploid ssRNA genome RNA genome in a virion is converted into

nto a DNA copy inside a cell by reverse transcription The DNA copy of the virion genome is permanently integrated into host chromosomes as a provirus (hence, a provirus is the DNA copy of a viral genome that has integrated itself into the host cell genome) The provirus directs synthesis of new RNA genome and mRNAs to make proteins to form new virions Virus particles assemble and bud from cellular membranes without killing the host cell Components of the retroviral virion: GAG proteins are structural Matrix (MA): located between Env and core Capsid (CA): main core protein Nucleocapsid (NC): binds RNA POL proteins are enzymatic (and are the target of antivirals) Protease (PR) cleaves viral polyproteins into active subunits (cleaves Gags) Reverse transcriptase (RT) converts RNA to DNA Integrase (IN) inserts DNA copy into host chromosome to make the provirus ENV proteins function in fusion; exists as a trimer that forms spikes + blobs on the surface of the virus Surface blob (SU) binds to receptor Transmembrane anchor/stick (TM) holds Env in plasma membrane and virion membrane Retroviruses are classified as either simple or complex Simple: mostly in animals (rous sarcoma virus, murine mammary tumor virus, moloney leukemia virus, mason-phizer monkey virus), the exception being XMRV that has been recently discovered Complex: HTLV-1, HIV-1, HIV-2 1. Know the basic steps of the retroviral life cycle. 1) attachment of virus to cell surface Involves Env fusuion proteins: surface blob and transmembrane stick 2) viral entry/genome uncoating Entry for HIV-1: Primary receptor is CD4. HIV-1 also uses a co-receptor, either CCR5 or CXCR4 Binding of HIV-1 to CD4 and co-receptor causes exposure of a fusion peptide, which promotes the mixing of cell and viral lipid membranes Side note on co-receptors:

CCR5 is more commonly transmitted. CXCR4 evolves years after infection, are more fusogenic, and associated with late stages of disease and progression of AIDS. People who lack CCR5 (via a naturally occurring polymorphism called CCR5R32) are highly resistant to infection! A drug called Maraviroc acts as a CCR5 inhibitor. 3) reverse transcription of viral genome RNADNA Reverse transcription is primed by host tRNA that has homology and can bind to the 5 end of the viral ssRNA genome The RNA has incomplete LTRs. However, reverse transcription produces a DNA copy that RECREATES complete LTRs at both ends! It is this complete version of DNA that gets integrated into the host genome in the next step. 4) genome integration into cellular chromosome, forming the provirus Integrase trims off two bases from each end of the DNA copy. It also makes staggered cuts in the upper and lower DNA strands of the host DNA. It then ligates the viral DNA onto the host DNA. Host cell DNA repair enzymes close the gaps. FYI: there is a characteristic 5 base pair duplication of host sequenes of either side Another FYI: HIV-1 integration is random but favors regions with active genes. 5) transcription of proviral genes DNARNA As mentioned before, the primary RNA transcript produced by regulation vi LTRs serves a dual function: as new RNA genomes as well as mRNA template. 6) virion assembly/release/maturation Gag and Gag-Pol gets to the plasma membrane by myristic acid present on their matrix proteins. Gag and Gag-Pol multimerize to provide the driving force for assembly and budding from the plasma membrane Env is a normal membrane protein that gets to the membrane via the conventional secretory pathways (ER, Golgi, etc) Genomic RNA is incorporated into the virion via nucleocapsid NC protein recognizing Once the virion is released, protease is activated, and it cleaves the polyproteins. That allows for the virion to mature.

2. Understand why retroviruses can cause cancer and their contribution to understanding oncogenes. Retroviruses can cause cancer via three mechanisms: 1) non-acute transforming viruses switch on the host oncogene. Think of it as a bad luck integration event causing insertional oncogenesis, where the retrovirus integrates near a cellular proto-oncogene, and the LTRs subsequently trans-activate its expression. 2) acute transforming viruses actually carry proto-oncogenes called v-onc (i.e. src gene in rous sarcoma virus). 3) HTLV-1 specifically has a gene called tax that produces a protein that transforms cells

3. Explain how retroviruses express their genes; understand basis for common antiretroviral drugs. How does gene expression work in retroviruses? LTRs (long terminal repeats) are responsible for producing ONE primary RNA transcript that serves as both the RNA genome that will be packed into new virions AND the mRNA encoding Gag, Pol, and Env. 5 LTR acts as the promoter. The enhancers within 5 LTR determine which cell types the virus can infect (i.e. HIV-1s 5 LTR has binding sites for transcription factors expressed in human T cells) U3 is located in 5 LTR and has enhancer/promoter elements that drive transcription 3 LTR acts as the poly(A) tail Since only one RNA transcript is made, additional RNA modification mechanisms are needed to produce all eight proteins of the retroviral virion: ribosomal frameshifting produces Gag-Pol Splicing makes Env Side note: splicing in HIV is even more complex, with differential splicing mech Precursor polyproteins are cleaved packaging signal incorporates viral RNA into particles that are recognized by the nucleocapsid proteins on Gag Antiretroviral drugs target processes that are unique to the retroviral life cycle. More specifically, they target enzymatic Pol proteins: Reverse transcriptase inhibitors (AZT, nevirapine) Protease inhibitors (RItonavir) Integrase inhibitors (Raltegravir) Fusion inhibitors (Fuzeon) CCR5 antagonists (Maraviroic) 4. Know why retroviruses make good gene therapy vectorsand potential downsides. Therapeutic retroviruses are engineered by: Removing the retroviral genes and adding therapeutic genes in their place Removal of retroviral genes (gag, env, pol) prevents the virus from replicating after delivering the gene! Benefits: Retroviruses make great therapy vectors because the DNA copy of their genome is integrated permanently into the host cell. The integration is stable and persists.

Downsides: while removal of retroviral genes prevents the therapeutic virus from replication, insertional oncogenesis can still occur!! In some cases, the vector integrates next to a cellular proto-oncogene called LMO-2, which leads to malignant transformation.