HIV: Virology, Genomics & Molecular Detection

HIV: Virology, Genomics & Molecular Detection Laboratorio de Biología Molecular Facultad de Medicina UASLP CA Garcia Sepúlveda MD PhD

Taxonomy Virus classification is based on: phenotypic characteristics, morphology, nucleic acid type, mode of replication, host organisms, & the type of disease they cause. A combination of two main schemes is currently in widespread use for the classification of viruses. - Baltimore Classification - International Committee on Taxonomy of Viruses Baltimore Classification (David L. Baltimore), American biologist, 1975 Nobel Prize "for discoveries concerning the interaction between tumour viruses and the genetic material of the cell." Also NF-Kβ, and recombination activating genes RAG-1 and RAG-2.

Taxonomy Baltimore Classification Scheme Based on the method used for viral mRNA synthesis. Seven groups depending on: nucleic acid (DNA or RNA), strandedness (ss or ds), Sense (positive or negative), and method of replication. Those in a given category will all behave in a similar fashion

Taxonomy Baltimore Classification Scheme Group I: dsDNA viruses Adenoviruses, Herpesviruses, Poxviruses, etc Variola HSV Polyoma

Taxonomy Baltimore Classification Scheme Group II: ssDNA viruses (+)sense Parvoviruses Parvo

Taxonomy Baltimore Classification Scheme Group III: dsRNA viruses Reoviruses Rotavirus

Taxonomy Baltimore Classification Scheme Group IV: (+)ssRNA Picornaviruses Togaviruses Equine Encephalitis Poliovirus Rubella

Taxonomy Baltimore Classification Scheme Group V: (-)ssRNA viruses Orthomyxovirus Rhabdovirus Orthomyxo Rhabdo

Taxonomy Baltimore Classification Scheme Group VII: dsDNA-RT integrating viruses Hepadnavirus HBV

Taxonomy Baltimore Classification Scheme Group VI: (+)ssRNA-RT viruses with DNA intermediate Retrovirus HIV-1

Taxonomy International Committee on Taxonomy of Viruses Orders Unassigned Families Retroviridae Subfamilies Orthoretrovirinae Genera Lentivirus Species

Taxonomy International Committee on Taxonomy of Viruses Family Subfamily Genus Species Alpharetrovirus Betaretrovirus Deltaretrovirus Epsilonretrovirus Gammaretrovirus Lentivirus Spumavirus Bovine leukemia virus Primate T-lymphotropic virus 1 Primate T-lymphotropic virus 2 Primate T-lymphotropic virus 3 Bovine Immunodef virus Caprine arthritis encephalitis virus Equine infectious anemia virus Feline immunodeficiency virus Human immunodeficiency virus 1 Human immunodeficiency virus 2 Puma lentivirus Simian immunodeficiency virus Visna/maedi virus Orthoretrovirinae Spumaretrovirinae Retroviridae

Retrovirus Key Features A. Human viruses associated with tumors, leukemias & immunodeficiencies. B. Common genetic organization and strategy - major differences lie in regulatory complexity. C. Genome consists of 2 copies of (+) stranded RNA. D. RNA converted to DNA by a bizarre mechanism followed by chromosomal integration (reverse transcription) . E. Integrated DNA co-linear with viral DNA.

Central Dogma Postulated by Francis Crick in 1958 and restated in 1970. 3x3 3 Biopolymers: DNA, RNA y polipeptides. 3 Modalities general, special and unreported. 3 Directions for each modality.

Central Dogma 3 General Directions: DNA to DNA (Replication). DNA to a RNA (Transcription). RNA to AA (Translation). 3 Special Directions: RNA to RNA (RNA Replication). RNA to DNA (Reverse transcription). DNA to AA (Direct translation, in vitro only). 3 Unreported Directions: AA to DNA (Intelligent evolution). AA to RNA (Intelligent evolution). AA to AA (prions).

Retrovirus • Examples • HTLV-1, HTLV-2… and HTLV-5 include members that can immortalize and transform target cells. • They are fast acting & cause sarcomas and leukemias through protooncogenes (±35) which cause disregulation of cell growth. • - Hormones, growth hormone receptors, protein kinases, GTP-binding proteins & nuclear DNA binding proteins.

Retrovirus Examples HIV-1 and HIV-2 include members that are slow viruses and associated with neurologic and immunosuppressive disease. HIV is a slow cytocidal virus with exquisite tropism for CD4+ expressing cells and macrophages. It is the loss of CD4+ cells which destroys helper and delayed type hypersensitivity functions of the immune response. Human foamy virus (spumavirus) not associated with disease.

Morphology Primate lentiviruses have a distinct morphology and can induce syncytia during productive infections. Electron micrograph showing HIV-1 particles in the process of budding from an infected cultured human PBMC, and several mature virions containing the characteristic conical/bullet-shaped nucleoid.

HIV Lentiviral member of the retrovirus family. Formerly:Human T-lymphotropic virus-III (HTLV-III), Lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Transmitted by blood, semen, vaginal fluid, pre-ejaculate and/or breast milk. Present in these body fluids both as free virus particles and as viral particles within infected immune cells. Four major routes of transmission are unprotected sexual intercourse, contaminated needles, breast milk, and transmission from an infected mother to her baby at birth.

Morphology HIV is different in structure from other retroviruses (spherical) Diameter of about 120 nm. 60 times smaller than a red blood cell, but very large for a virus. Contains two copies of positive single-stranded RNA that codes for the virus's nine genes.

Morphology HIV is different in structure from other retroviruses (spherical) Diameter of about 120 nm. 60 times smaller than a red blood cell, but very large for a virus. Contains two copies of positive single-stranded RNA that codes for the virus's nine genes. Envelpoed by a matrix protein shell. Protected by a conical capsid. Stabilized by a nucleocapsid.

Morphology Organization of a mature human immunodeficiency virus (HIV)-1 virion. Plasma membrane envelope Surface envelope glycoprotein Transmembrane envelope glycoprotein Matrix protein (p17) Capsid (p24) Protease (p11) Reverse Transcriptase (p66/p51) Nucleocapsid (p7) Viral RNA genome Integrase (p31) Vi

Morphology The single-stranded RNA is tightly bound to nucleocapsid proteins, p7 and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase.

Morphology Envelope consists of a cap made of three molecules called glycoprotein (gp) 120, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope. This complex enables the virus to attach to and fuse with target cells.

Morphology Functional relevance

Morphology Structural integrity of mature viral particle kept by matrix protein, capsid, nucleocapsid and envelope.

Morphology Homing and attachment involve gp120 and gp41.

Morphology Fusion involves both matrix proteins (p17) and the envelope

Morphology Reverse transcription requires Reverse Transcriptase (p66/p51)

Morphology Integration requires intgrase (p31) and ribonuclease.

Morphology Expression and assembly requires protease (p11).

Genome Genomic organization of simple and complex retroviruses. Simple retrovirus Moloney murine leukemia virus (MuLV) contains: Long terminal repeat (LTR) sequences provide transcriptional regulatory elements. Gag encodes structural proteins of the virus. Pol encodes enzymes involved in reverse transcription / integration. Env encodes the virion surface glycoproteins.

Genome Genomic organization of simple and complex retroviruses. Simple retrovirus Moloney murine leukemia virus (MuLV) contains: Long terminal repeat (LTR) sequences provide transcriptional regulatory elements. Gag encodes structural proteins of the virus. Pol encodes enzymes involved in reverse transcription / integration. Env encodes the virion surface glycoproteins. Group Specific Antigen Polymerase Envelope

Genome Since HIV has a more complex life cycle than simple retroviruses such as MuLV one can expect a more complex genome. HIV can control its replication in a more complex fashion. HIV genome length is of 9749 bases (average for retroviruses). Where does the difference lie, where is the rest of the complexity encoded?

Genome DUAL LAYER DVD ! Makes use of overlapping reading frames (ORFs). ORF: Open Reading Frame 1: ATG CAT GCA TGC ATG 2: TGC ATG CAT GCA TGC 3: GCA TGC ATG CAT GCA Same genome length, higher density!

Genome HIV-1 Encodes for more proteins than theoretically possible with a single ORF. Overlapping genes (such as ENV, TAT and REV use different ORFs of the SAME genomic region. Genes in different ORFs (REV) can be split by other genes (TAT). HIV genome has nine open reading frames that code for 19 proteins. Some of these EXTRA proteins are involved in transcriptional regulation.

Genome RNA genome consists of at least 7 structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and tev) encoding 19 proteins.

Genome Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles.

Genome Gag region is 2000 bp and codes for the core structural proteins (matrix, capsid & nucleocapsid). Initial transcript size is p53, final products are p18, p24 and p15.

Genome Pol region is the longest at 2900 bp and codes for the enzymes. Initial transcript size is p160. Final cleavage products are p10 (protease), p66 (RT) and p32 (integrase).

Genome Env region is the shortest structural gene at 1800 bp and codes for the surface and transmembrane glycoproteins. Initial transcript size is p160. Final cleavage products are gp120 and gp40.

Genome The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.

Genome The two Tat proteins (p16 and p14) are transcriptional transactivators for the LTR promoter acting by binding the TAR RNA element

Genome The Rev protein (p19) is involved in shuttling RNAs from the nucleus and the cytoplasm by binding to the RRE RNA element.

Genome The Vif protein prevents the action of APOBEC3G (a cell protein which deaminates DNA:RNA hybrids and interferes with the Pol protein).

Genome The Vpr protein (p14) arrests cell division at G2/M.

Genome The Nef protein (p27) downregulates CD4 (the major viral receptor), as well as the MHC class I and class II molecules.

Genome The Vpu protein (p16) influences the release of new virus particles from infected cells.

Reverse Transcription

HIV: Virology, Genomics & Molecular Detection