Retroviridae • Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. • This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders , and cause degenerative and neurological syndromes. Retrovirus Virions Thin Section EM of Some Retroviruses Type A Type B (eccentric) MMTV TC(tl)Type C (central) ALV,RSV Type D (bar) Lentivirus (cone) HIV Retrovirus structure • Retrovirus virions are 80-120 nm in diameter , have spherical morphology, a phospholipid envelope with knobs • Contain around 2000 molecules of nucleocapsid (NC) protein that bind to the two copies of (+) strand RNA genome •Retroviral ribonucleoproteins are encased within a protein shell built from the capsid protein to form an internal core, which can have different shapes and has a conical shape in HIV Retrovirus Structure and Function Figure 10.23a Retrovirus Classification Family: Retroviridae Genus Features Examples 1. Alpharetrovirus Simple, Avian leucosis virus, RSV Onco 2. Betaretrovirus Simple, Mouse Mammary Tumor Onco Virus 3. Gammaretrovirus Simple, Murine leukemia virus Onco ((yy)Moloney, Harvey) 4. Deltaretrovirus Complex, Bovine Leukemia, Human Onco T Cell Leukemia (HTLV) 5. Epsilonretrovirus Complex, Walleye Dermal Sarcoma Onco 6. Lentivirus Complex HIV, Visna, EIAV 7. Spumavirus Complex Simian Foamy Virus • RNA virus, 120nm in Family : Retroviridae diameter Subfamily : Lentivirus • Envelope gp160; gp120 & gp41 • Icosahedral symmetry • NlNucleocapsid Outer matrix protein (p17) MjMajor capsid protitein (p24) Nuclear protein (p7) • DilidDiploid RNA wihith several copies of reverse transcriptase Retrovirus replication cycle 1. Attachment of the virion to a specific cell surface receptor 2. Penetration of the virion core into the cell 3. Reverse transcription within the core structure to copy the genome RNA into DNA 4. Transit of the DNA to the nucleus 5. Integration of the viral DNA into random sites in cellular DNA to form the provirus 6. Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template 7. Processing of the transcripts to genome and mRNAs 8. Synthesis of virion proteins 9. AblAssembly and buddi ng of viiirions 10. Proteolytic processing of capsid proteins Retrovirus life cycle: Electron micrograph of HIV budding HIV Structure surface transmembrane matrix protein capsid protein nucleocapsid protein RT Integrase protease Retroviral Proteins • gag, pol, and env – Gag protein proteolytically processed into • MA (matrix) • CA (capsid) • NC (nucleocapsid) – Pol protein encodes enzymes • PR (protease) • RT ((pReverse Transcriptase which has both DNA pypolymerase and RNase H activities) • IN (Integrase) – Env protein encodes • SU sur face g lycoprote in • TM transmembrane protein • “Accessory” genes (in Complex Retroviruses) - regulate and coordinate virus expression; function in immune escape • Oncogene products (v-Onc, in Acutely Transforming Retroviruses) - produce transformed phenotype Regulatory proteins: Tat • HIV LTR functions as a promoter in a variety of cell types in vitro • It includes an enhancer sequence that binds a number of cell type specific transcription activators Stimulation of transcription by HIV‐1 Tat protein: • Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site • Production of the Tat protein allows transcription complexes to synthesize full length RNA • Binding of Tat to TAR together with the cyclin T subitbunit of TkTak ldleads to stimul ati on of phosphorylation of the largest subunit of RNA polymerase II • As a result, the transcri pti onal complexes become competent to carry out transcription Regulatory proteins: Rev • Rev Protein is an RNA binding protein that recognizes a specific sequence within the structural element in env called the Rev‐responsive element (RRE) HIV accessory proteins Nef protein: • Translated from multiply spliced early transcripts • myristylated at its N‐terminus and anchored to the inner surface of the plasma membrane • Nef deleted HIV and SIV are much less pathogenic in vivo • Nef downregulates expression of CD4 by enhancing endocytosis • Can activat e CD4+ T lhtlymphocytes by modulating signaling pathways HIV accessory proteins Vif Protein: • Viral infectivity factor • Accumulates in the cytoplasm and at the plasma membrane of infected cells • Mutant viruses lacking the vif gene were less infectious and dfdefec tive in some way • vif‐defective virions enter cells, initiate reverse transcription, but do not produce full‐length double stranded DNA • vif inhibits antiviral action of a cytidine deaminase, which is synthesized in nonpermissive cells •This enzyme deaminates deoxyyycytidine to deoxyuridine and leads to endonucleolytic digestion or G to A transitions HIV Genome • 9200 nucltidleotides (HIV‐1): • env ‐ gp160 (gp120:outer membrane part, gp41: transmembrane part) • gag core proteins – p55, p17 and p24 • pol –p66 (protease), p31,p51 (integrase/endonuclease) EARLY Accessory Genes LATE tat ‐ trans activator of transcription vif ‐ viral infectivity factor vpr‐ viral protein R rev‐ regultlator of viliral protitein expression vpu‐ viliral protitein U nef –negative regulatory factor vpx –HIV ‐ 2 TAT and REV are essential for HIV replication Subtyping of isolates • RFLP Analysis • Nucleotide sequence analysis • SbSubtype specific genetic probes Genetic variation in HIV Mutations occur 65 times more than that observed in Influenza virus HIV‐1 –Three groups HIV‐ 2 –eight M (Maj or) subtypes A‐H O (()Outlier) N (New) Subtypes of HIV Subtypes of HIV‐1 – M • 10 Subtypes/Clades/Genotypes Designated A‐K Differ in Geographical distribution and major mode of Transmission Africa ‐ ACA,C , D US and Western Thailand ‐ E, B Europe ‐ B IdiIndia ‐ C Transmission • Sexual Activity – both homosexual and heterosexual. – Women are more easily infected through intercourse than are men. • Injecting drug use • Tainted blood transfusions – extremely rare since 1986. • Transmission across the placental barrier, during the birth process, or through mother’s milk. HIV Transmission • HIV enters the bloodstream through: – Open Cuts – Breaks in the skin – Mucous membranes – Direct injection HIV Transmission • Common fluids that are a means of tiitransmission: – Blood – Semen – Vaginal Secretions – Breast Milk HIV in Body Fluids Blood Semen 18,000 11,000 Vaginal Fluid Amniotic 7,000 Fluid 4,000 Saliva 1 Average number of HIV particles in 1 ml of these body fluids ENTRY OF HIV INSIDE T CELL HIV ‐ Life History CCR5 ‐ the co receptor HIV chemokine Mutant CD4 CCR5 CCR5 CD4 CD4 CCR5 macrophage Virion interaction with CD4 receptor and CXCR4 co‐ receptor HIV and AIDS The cellular and immunological picture - The course of the disease July 2008 e Serology Laboratory diagnosis of HIV infection • Direct demonstration of infective agent - Virus isolation- virus culture -Antigen detection- P24 detection - viral nucleic acid detection- PCR • Indirect demonstration of infective agent - Anti -HIV antibody detection Types of HIV Diagnostic Tests HIV Antibodies HIV‐1 RNA HIV p24 Antigen Most Common Test for Used for Acute HIV and Rarely Used. Future use: 4th Established Infection Indeterminate WB Generation EIA Specimens to be collected for AibddAntibody detect ion • Blood / Serum / Plasma • Saliva / Urine Note! Specimen requiring storage before shipping to lab: -No longer than 7 days at 4◦C - No longer than 3 days at RT - For longer period serum or plasma must be separated from clot or cells and stored at -20 ◦C - For PCR testing samples need to be processed within 48 h 0f collection. Initial and Supplemental HIV Tests • Initial Test - Enzyme Immunoassay (EIA) - Chemil um inescent Immunoassay (CIA) • Supplemental Tests - Western blot - IdiIndirect IflImmunofluorescence A(IFA)Assay (IFA) - Qualitative HIV-1 RNA Generation of EIA Tests First Second Third *Fourth *Not US FDA‐approved as of 10/1/09 UdilUses crude viral UbitHIVUses recombinant HIV DtDetec ts IMIgM an dIGd IgG De tec ts HIV an tibo dies lysate antigens or peptides in “Sandwich” EIA and p24 antigen Rapid tests Advantages: 1. Quick 2. Easy to perform 3. No sophisti ca te d ins trumen ts are requidired 4. Can be done on single sample Disadvantages: 1. Costly 2. Tedious if large no. samples have to be tested at one time FACTORS KNOWN TO CAUSE FALSE POSITIVE HIV ANTIBODY TEST RESULTS Passive immunization: receipt of gamma globulin or immune globulin (as pppyrophylaxis against infection which contains antibodies) Leprosy Tuberculosis Mycobacterium avium Systemic lupus erythematosus Renal (kidney) failure Hemodialysis/renal failure Alpha interferon therapy in hemodialysis patients Flu Flu vaccination Herpes simplex I & Herpes simplex II Upper respiratory tract infection (cold or flu) Recent viral infection or exposure to viral vaccines Malaria FACTORS KNOWN TO CAUSE FALSE POSITIVE HIV ANTIBODY TEST RESULTS Pregnancy in multiparous women Hepatitis B vaccination Tetanus vaccination Organ transplantation Renal transplantation Anti‐lymphocyte antibodies Anti‐collagen antibodies Serum‐positive for rheumatoid factor, antinuclear antibody (both found in rheumatoid arthritis and other autoantibodies) Autoimmune diseases lupus erythematosus, scleroderma, connective tissue disease Acute viral infections, DNA viral infections Malignant neoplasms Confirmatory Tests Western Blot, Line immunoassay • WB use antigens from whole virus lysates electrophoretically transferred