HIV

HIV causes AIDS (aquired immune deficiency syndrome) Infects specific cells of the immune system CD4+ helper T cells Macrophages (scavenger APC cells) Follicular dendritic cells Symptoms Profound immunosuppression Opportunistic infections Specific malignancies or cancers Sometimes central nervous system degeneration

HIV – a retrovirus Lentivirus subfamily of retroviruses Genome is RNA Two identical strands of single stranded RNA ~ 9.2 kb in length The RNA is of the positive sense (the strand is the same sense as mRNA) Although the RNA is of the positive sense, expression of the genome requires that the virus is copied into cDNA and that the cDNA is integrated into the host cell DNA

The structure of HIV Envelope A lipid bilayer derived from the host cell plasma membrane as the virus exits the cell. The envelope surrounds the protein coat of the virus. Embedded in or associated with the envelope are several virally encoded proteins gp (glycoprotein) 120 gp41 gp120 and gp41 are synthesized as a precursor protein (gp160) that gets cleaved into gp 120 and gp 41 which remain associated with each other through disulfide bonds. Only gp 41 is embedded in the envelope.

HIV – gp120 and gp41 gp120 gp41 CD4

Structure of HIV Matrix protein – p17 (gag encoded protein found on the inner surface of the envelope)

The structure of HIV Capsid The outer protein coat of of the virus Composed repeating units of the protein p24 (called capsomers) Nucleocapsid Consists of the capsid plus the viral RNA which is associated with several proteins Reverse transcriptase (has RNAse H activity in addition to a polymerase activity) Integrase Protease

HIV structure

Genome of HIV

Genome of HIV GAG encodes Pol encodes Env encodes Capsid structural protein (p24) Matrix protein (p17) An RNA binding nucleocapsid protein Pol encodes Reverse transcriptase Integrase Protease Env encodes Envelope glycoproteins – gp120 and gp41 Several others smaller genes as summarized on previous slide

HIV attachment There is recent evidence suggesting that a host cell protein incorporated into the virus during assembly, cyclophilin A, serves to initially bind the virus to the low affinity receptor heparin sulfate. This is followed by the binding of the viral ligand, gp120, to CD4. CD4 is found on T helper cells, macrophages, and glial cells. The binding of gp120 to CD4 results in a conformational change of gp120 which then binds to a chemokine coreceptor, CXCR4 on T lymphocytes, or CCR5 on macrophages. CD4 is involved in the recognition of MHC antigens on B lymphocytes and functions in the immune response in which antibodies are made.

HIV attachment Chemokine receptors are receptors of chemical messages involved in cellular communication. Individual HIV strains are classified as being either lymphotropic or macrophageotropic based on which chemokine receptor the virus recognizes. Rare individuals who are resistant to HIV may have mutations in their chemokine receptors.

HIV binding to CD4 and chemokine receptors gp120 gp41 CD4

HIV penetration Binding of gp120 to its receptor and coreceptor causes a conformational change in gp41 Gp 41 mediates fusion of the viral envelope to the plasma membrane of the host cells via the activity of the fusion peptide This results in the release of the nucleocapsid into the cytoplasm.

HIV penetration

Fusion at the cell membrane (pH independent)

Replication of HIV Reverse transcriptase uses the RNA, and with RNAse H removal of viral RNA, the first strand cDNA, as templates to make double stranded DNA which circularizes and moves into the nucleus. Viral integrase participates in integrating the ds DNA into the host cell DNA. This is a random integration, but integration is absolutely required for viral replication. Note that reverse transcriptase and the integrase come into the cell as part of the viral nucleocapsid.

Replication of HIV Translation of the early gene mRNA transcripts occurs followed by translation of the late gene mRNA transcripts. HIV is unique among retroviruses in having several genes that encode proteins that regulate viral transcription. These regulatory genes are the first genes to be expressed.

Replication of HIV Next the structural genes are expressed. The gag genes and pol genes are expressed as polyproteins that get cleaved by the VIRAL protease into the individual gag and pol proteins. The env gene is also expressed as a polyprotein, gp160, that is cleaved into gp120 and gp41 by CELLULAR proteases.

Replication of HIV Insertion of Env proteins into host cell membrane Gp 120 and gp 41 remain bound to each other through disulfide bonds They are transported to and inserted into the plasma membrane of the infected cell. Host cells proteins are pushed aside resulting in patches of plasma membrane that have been modified by the addition of viral proteins.

Replication of HIV During late gene expression, viral genomic RNA is transcribed Once enough genomic RNA and viral structural proteins are made, assembly begins Assembly of the nucleocapsid occurs when full-length RNA genomic transcripts are complexed with the the gag and pol encoded proteins.

Replication of HIV The nucleocapsids acquire their envelope by budding from the host cell and taking the virally modified plasma membrane as their envelope. Thus, mature infectious virus is released. This may or may not result in lysis of the infected cell.

Progression of HIV Infection The initial target of HIV infection is the macrophage via the CCR5 “co”receptor, but the virus replicates very poorly in macrophages. Macrophages bring the virus to the lymphatic tissue where infection of CD4+ T cells occurs by HIV variants that can bind to the T cell CXCR4 coreceptor. These variants are generated via reverse transcriptase mistakes made during replication of the virus in the macrophage (RT has no editing functions).

An infected macrophage. Viral particles are within the vacuoles.

Progression of HIV Infection If the CD4+ T cell is inactive, the virus remains latent, but if the T cell is active, viral transcription using host cell enzymes begins. Viral mRNAs and genomic RNA are made. Initiation of transcription in T cells is linked to physiologic activation of the T cell by antigen or by cytokine stimulation.

Contributions to clinical immunodeficiency Lysis of CD4+ T cells caused by viral budding and/or env glycoprotein insertion or Endogenous gp120 prevents CD4 from being transported to the cell surface (newly made or recycled CD4) leading to cytopathic effects or Down regulation of MHC class I molecules cause NK cells to destroy the infected cell or other cytopathic effects of unintegrated viral RNA or DNA or syncytium formation (giant, multinulcleated cells formed by the fusion of the membranes of adjacent cells)

Syncytia formation

Contributions to clinical immunodeficiency Loss of CD4+ T cells by indirect effects Inhibition of maturation gp120 cross-linking of CD4 receptors priming cells for apoptosis Autoimmune destruction (cross reacting antibodies against gp120 or other viral proteins also react with self antigens on the T cell) Super-antigen effect – a super antigen binds to the exposed surfaces of MHC class II molecules on antigen presenting cells and to the variable region of the TCR to activate the cell non-specifically or cause anergy (absence of reaction to the antigen), or deletion of specific T cell subsets

Superantigens What is a superantigen? It causes non-specific stimulation of T cells leading to cytokine release and inflammation which leads to fever, hypertension and shock.

Contributions to clinical immunodeficiency Functional impairment of immune system Soluble gp120 blocks interaction of CD4+ T cells with Class II MHC on antigen presenting cells (APCs) Endogenous gp120 prevents CD4 from being transported to the cell surface. Impaired macrophage and natural killer cell function Destruction of architecture of lymph nodes

Contributions to clinical immunodeficiency Central nervous system damage Release of inflammatory cytokines from HIV-infected CNS macrophages Soluble gp120 may interfere with neurotransmitter action on neurons

Diagnosis of HIV infection ELISA – used for first screening Western blot – used to confirm a positive ELISA result RT-PCR – used for following viral load (viral concentration) during treatment Detects the presence of viral RNA by making a cDNA copy of the RNA and amplifiying that copy by PCR.

Treatment of HIV infection Nucleoside analogues such as AZT Prevent successful synthesis of cDNA by RT; act as chain terminators when incorporated in place of a normal nucleoside Non-nucleoside analogue RT inhibitors Bind non-competitively to RT Protease inhibitors Prevent cleavage of polyproteins required to make mature virus Combination therapy – helps prevent the development of resistant strains HAART - highly active anti-retroviral therapy (3 or more drugs in combination)

Treatment of HIV infection Anti-inflammatories are being considered Prevent destruction of the lymphatic architecture

How does the biology of this HIV unit relate to the biology of the HLA DQ-alpha unit?