Human Immunodeficiency Virus

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1 Human Immunodeficiency Virus 1. Identification of the AIDS Virus a) opportunistic infections observed in homosexual men (all had T4 helper cell depletion) -> termed Acquired Immune Deficiency Syndrome; Summer CDC got reports of unusual pneumonia (Pneumocystis carinii rare, usually harmless protozoan rarely associated with pneumonia), Kaposi s sarcoma (rare tumor of blood-vessel tissue associated with aging) in homosexual men in NY, LA, and SF b) infectious agent probable, possibly a virus such as CMV or EBV c) patients with persistant LymphAdenopathy Syndrome (LAS) had characteristics reported for HTLV (human T-cell leukemia virus) d) RT activity could be found in patients with LAS e) a retrovirus agent similar to HTLV was be isolated from AIDS-infected patients f) HTLV transforms lymphocytes whereas the AIDS virus lysed lymphocytes Spring

2 HIV g) HTLV is not the AIDS virus > HTLV-III (French LAV lymphadenopathy-associated virus) h) Does this virus cause AIDS? What are possible mechanisms for virus detection? How do they differ from AIDS surveillance? What is the real problem here? The virus was later reclassified as the Human Immunodeficiency Virus (HIV), a member of the lentivirus family (it is not oncogenic). Spring

3 Virus Distribution a) current distribution of HIV-infected adults (12/99) Africa 24.5 million South & Southeast Asia 6.7 million Latin America 1.4 million North America 0.9 million Europe 0.5 million Spring

4 HIV in Africa Spring

5 AIDS in the U.S. in 1998 Spring

6 Annual Number of Deaths in the U.S. Spring

7 HIV b) spread of virus: sexual contact, blood, mother to infant 1) infection by virus 2) infection via virus-infected cells c) hallmark of HIV infection is the decline in T4 helper cells Spring

8 d) HIV disease progression: HIV 1) initial acute infection (usually quite mild; flu-like) 2) gradual reduction in T4 cell counts 3) accumulation of opportunistic infections (these will eventually kill the patient, not the direct effect of HIV replication!) Spring

9 Table 5.6 Pathology and Opportunistic Infections of Patients with HIV/AIDS STAGE OF HIV/AIDS SYNDROME/SYMPTOMS TYPE OF INFECTIOUS AGENT ORGANISM CD4 + T-Cell count cells/µl Generalised lymphadenopathy Headache, fever, malaise Gneralized weight loss Shingles Viral Reactivation of herpes zoster Skin lesions Viral Molluscum contagiosum Basal cell carcinomas of skin Oral lesions Thrush Fungal Candida albicans Hairy Leukoplakia Viral Epstein-Barr virus Lung disease (Tuberculosis) Bacterial Reactivation of Mycobacterium tuberculosis CD4+ T-cell count <200 cells/µl (Syndromes in addition to those mentioned above) Microbial Infections Pneumonia Protozoan Pneumocystis carinii Disseminated toxoplasma Protozoan Toxoplasma gondii Severe diarrhea Protozoan Isospora belli Chronic diarrhea Protozoan Cryptosporidia Tuberculosis Bacteria Mycobacterium tuberculosis Bacteria Salmonella, Streptococcus, Hemophilus, Cryptococcus CNS disease Fungal Cryptococcus neoformans Viral Infections and Malignancies PML Viral JC polyomavirus Disseminated disease of Viral Genital herpes, cytomegalovirus lungs,brain, etc. B-cell lymphoma Viral Epstein Barr virus, HHV-8 Kaposi s sarcoma Viral HHV-8 Anogenital carcinoma Viral Human papilloma virus Other Syndromes Wasting disease???? Aseptic meningitis AIDS dementia complex Viral HIV encephalopathy Spring Abbreviations used: CNS = central nervous system; PML = progressive multifocal leukoencephalopathy; HHV-8 = human herpesvirus eight. Adapted from Coffin et al. (1997) Table 1 on page 597.

10 Two major glycoproteins: HIV Envelope Proteins a. synthesized as 160kD precursor that is processed by cellular protease b. gp41 is a transmembrane protein that contains an external domain required for fusion with target cells c. gp120 protrudes from envelope and binds to CD4; contains most neutralization epitopes Spring

11 HIV Pathogenesis T cells are the major site of infection but macrophages, dendricytes, monocytes have low levels of CD4 and may harbor HI--> infection of microglial cells may cause AIDS-related neurological problems Cell Types that can be infected by HIV: a) Hematopoietic/Immune cells: b) Brain/glial cells T cells Microglia B cells Glial cell lines Primary monocytes/macrophages Fetal neural cells Kuppfer cells Brain capillary endothelium Monocyte cell lines c) Others BM precursor cells Fibroblasts Dendritic cells Colon carcinoma cells Langerhans cells Liver sinusoid epithelium Osteosarcoma cells Spring

12 Fusion Process What are the coreceptor molecules? a. a-chemokine receptor - CXCR4, also known as fusin: 7-transmembrane receptor protein T-tropic viruses use this co-receptor; these strains infect primary and established CD4 + T cells b. b-chemokine receptor - CCR5: 7-transmembrane receptor protein M-tropic viruses use this co-receptor; these strains infect primary CD4 + T cells They represent 90% of the sexual transmission of HIV Spring

13 Chemokine Receptors Spring

14 Mechanisms for HIV Induced Cell Death HIV Action or, How to Kill a T Cell CD4 + I CD4 + T cell HIV infection and cell death due to viral propagation Infected T cell Uninfected T cell Synctium (Multinucleated Giant Cell) II GP120 expressed on surface of infected cell binds Spring 2002 to the CD4 receptor on uninfected cell. 420 The cells fuse and the resultant giant cell dies.

15 Mechanisms for HIV Induced Cell Death Antibodies Infected T cell III Natural Killer Cells and Cytotoxic T cells Infected T cells expressing GP120 on their surfaces, can be killed by a normal immune reponse by antibodies, NK cells or cytotoxic T cells Uninfected T cell Free GP120 from the blood stream shed from infected cells IV Free GP120 from the blood stream can bind to the CD4 molecule Spring 2002 and make a healthy T cell appear to be infected by the body's 421 immune system and subsequently killed by NK, T cells and antibodies

16 Autoimmunity Autoimmunity: HIV disturbs the balance of the immune system and autoimmune disorders accompany the virus. a. Over-reacting immune system: autoantibodies to a large number of normal cellular antibodies b. T-cell dysregulation c. B-cell proliferation with resultant antibody production: probably results for IL-6 production by HIV infected cells or reactive macrophages. d. Molecular mimicry: Viral gene(s) and/or gene product(s) sharing sequence homology with a normal cellular component 1. Sometimes this can elicit immune responses that cross react with normal cells. 2. Ex: cross reactive antibodies that recognize a platelet glycoprotein and HIV gp120 cause a disease called thrombocytopenia. e. Anti-idiotype antibodies (AIA): antibodies to anti-antigen antibodies may mirror images of the epitope against which the initial antibody was produced 1. Ex: Antibodies to gp120 antibodies might induce autoantibodies against CD4. 2. It is unclear if AIA play a significant role in HIV biology. General conclusions on potential importance of autoimmune responses (see fig). 1. Autoimmune responses may be potential cofactors in HIV pathology 2. Illustrates a potential danger in vaccination with HIV proteins Spring

17 Autoimmunity Spring

18 Antibody Response Spring

19 Steps of HIV Pathogenesis a. Virus initially enters an individual primary by infecting either activated T cells, resident macrophages, or mucosal cells in the bowels or uterine cavity b. In the initial days following infection, high level of virus will take place in the lymph nodes and will be reflected by high level of antibodies to p24. c. CD8+ cell numbers will rise and within 1 month virus antigen titers will reduce substantially. d. Over the ensuing months to years, CD8+ cell number remains slightly elevated. Virus replication still persists in the lymph nodes e. CD4+ cell usually rise to near normal levels 3-4 months after primary infection f. CD4+ cells then decrease steadily during the persistent period (for unknown reasons) g. The individual begins to develop symptoms when CD4+ cells drop below 300 cells per ul and HIV in blood increase. h. At the same time there is a reduction in antiviral CD8+ cells i. Symptomatic infection develops, the virus has characteristics distinct from the virus recovered soon after the infection: 1. Enhanced cellular host range 2. Rapid kinetics of replication 3. CD4+ cell cytopathicity j. More virulent variants of the virus replicate to higher levels and destroy a large number of CD4+ cells. This eliminates the ability of the body to mount any immune response to infection k. In the terminal stage, both CD4+ and CD8+ continue to decrease Spring

20 HIV Genome Proviral DNA LTR LTR ORFs Frame 3 Frame 2 Frame 1 gag pro tat vpu vpr pol env vif rev rev tat nef Genomic RNA (gag mrna) (gag-pro-pol mrna) CAP TAR RRE An Other mrnas vif mrna CAP An vpr mrna CAP An tat mrna CAP An rev mrna CAP An vpu, env mrna CAP An nef mrna CAP An kb Spring

21 Table 5.4 The HIV Proteins PROTEIN mrna SIZE kd POST-TRANSLATIONAL FUNCTIONS MODIFICATIONS gag genomic RNA p25 (CA) none Capsid structural protein p17 (MA) myristylated at 2Gly Matrix protein p7? RNA-binding protein p2? RNA binding protein pro genome RNA frameshifted p10 (PR) Viral protease, processes gag proteins pol genome RNA frameshifted p66/p51 RT Heterodimer, p51 lacks RNase H domain present in p66 Reverse transcriptase vif vif mrna p23 viral infectivity factor, essential for spread in macrophages vpr vpr mrna p15 associates with p7 Augments replication tat tat mrna p14 Required for replication transactivates RNA synthesis, binds to TAR RNA rev rev mrna p19 Regulates splicing/rna transport; binds RRE element and facilitates env translation vpu vpu/env mrna p16 phosphorylated on Ser Helps in virion assembly and release, dissociates gp160/cd4 complex env vpu/env mrna gp 120 (SU) 24 sites for N-linked glycosylation Surface glycoprotein, mediates cellular attachment gp 41(TM) 7 sites of N-linked glycosylation Transmembrane glycoprotein nef nef mrna p27 myristylated at Gly-2, phosphorylated at Tyr-15 Homodimer, causes pleiotropic effects, including downregulation of CD4 Adapted from Levy (1994) p. 8. Spring

22 HIV Relatives SIV: simian immunodifficiency virus a. discovered by analyzing sera from macaques: they had antibodies that cross-reacted with HIV proteins (gag) b. virus is 50% related to HIV-1 at the nucleotide level c. not a direct precursor to HIV-1 HIV-2 : human mmunodifficiency virus - 2 a. discovered by analyzing sera from high-risk humans from West Africa -> cross-reacted with both gag and env proteins of SIV b. more closely related to SIV than to HIV-1 c. produces symptoms similar to HIV-1 although less pathogenic Spring

23 HIV Replication I. Virus attachment and fusion a. gp120 interacts with domain 1 of CD4 (4 Ig-like domains) b. The interaction causes a conformation change that exposes a binding site for thechemokine coreceptor, c. This interaction results in exposure of the TM fusion peptide and a trimeric coiled coil transition that facilitates membrane fusion II. Virus-cell fusion a. HIV enters through a ph-independent route b. HIV entry (and subsequent replication) down regulates CD4 expression on the cell surface III. Synthesis of viral DNA and integration a. Once the virus has entered the cell through the viral core, synthesis of proviral DNA takes place within the first six hours after infection in the cytoplasm b. Many current HIV drugs are nucleotide analogs that block synthesis of viral DNA c. Integration of the provirus into the host cell chromosome appears random and is mediated by the viral encoded integrase protein; once integrated, the viral DNA remains permanently associated with the host genetic material Spring

24 HIV Replication VI. Intracellular control of HIV replication a. The big question: What controls the HIV latency period? b. Review of general retrovirus transcription c. HIV has many different mrnas which result from alternative splicing c. The initial rate of transcription from the HIV 5' -LTR is controlled by host cellular proteins (transcription factors) 1. In most cell types HIV LTR promoter is weak 2. Promoter activity is requires cellular transcription factors (see figure): a. NF-kB, NFAT, AP-1, SP-1 b. Viral ' tat' protein is also required 3. The ability of HIV to replicate in particular cell type may be dependent of the relative abundance of the required cellular factors a. Ex: Macrophages vs. T cells b. Cellular proteins can regulate availability of TFs 1. Ex: NF-kB is inhibited by IkB c. Cytokines can stimulate HIV expression via stimulation of NF-kB Spring

25 VII. HIV transcription is tightly regulated and split into two segments: a. Early mrnas correspond to the regulatory proteins tat, rev, and nef 1. The balance of these regulatory proteins play a key role in controlling cellular viral latency. 2. Tat: A viral transactivator is a viral encoded regulatory protein. a. Found in the nucleus b. Binds to a stem-loop structure (called ' tar' ) at the 5' end of the nascent RNA (see figure) c. Binding of tat increases the stability and frequency of the polymerase which are transcribing viral genes and permits synthesis of full-length RNAs d. The full-length viral RNA transcript contains multiple splicing sites which are utilized to produce multiple spliced viral mrnas 1. mrnas especially for rev and nef e. Binding of cellular proteins to Tat or Tar may play a role in regulating activity e. Tat is a positive regulator of transcription 3. Nef: A negative regulator a. Also found in the nucleus b. Nef suppresses HIV replication: mechanism is not clear 1. Could directly down regulate transcription from the LTR 2. Nef may inhibit NF-kB induction 3. Nef may have and anti-rev activity c. Nef may be acting through interactions with cellular protein (via phosphorylation) d. Expression of Nef may be critical to maintain HIV cellular latency Spring

3. Nef: A negative regulator a. Also found in the nucleus b. Nef suppresses HIV replication: mechanism is not clear 1. Could directly down regulate transcription from the LTR 2.

26 3. Nef: A negative regulator a. Also found in the nucleus b. Nef suppresses HIV replication: mechanism is not clear 1. Could directly down regulate transcription from the LTR 2. Nef may inhibit NF-kB induction 3. Nef may have and anti-rev activity c. Nef may be acting through interactions with cellular protein (via phosphorylation) d. Expression of Nef may be critical to maintain HIV cellular latency 4. Rev: Plays a key regulatory role in balancing Tat and Nef and the production of unspliced mrnas a. Rev controls expression of late gene products and progeny viral RNAs b. In the absence of Rev, only small multiply spliced RNAs accumulate in the cytoplasm c. In the presence of Rev, unspliced and singly spliced viral messenger RNAs can accumulate in the cytoplasm d. Rev is also located in the nucleus e. Rev binds to a complex stem-loop structure within the envelope coding region (see figure) 1. Binding region termed the ' RRE": rev responsive element f. The mechanism of Rev activity is unclear 1. Binding of Rev to RRE may directly inhibit cellular splicing activity 2. Binding of Rev to RRE may permit the viral RNA to bypass the splicing machinery Spring Conclusion: Both viral and cellular factors, working together, affect HIV replication.

27 b. Late regulatory genes: facilitate virus release and increase virus particle infectivity 1. Vif: made from a singly spliced mrna which accumulates late in infection a. Deletion of vif results in release of the virus from the cell which are poorly infectious b. Vif may act as a cysteine protease to clip the carboxy terminus of the envelope protein gp Vpu: Facilitates the export of virus particles from the cell a. Vpu is translated from the singly spliced env mrna. b. Acts as a bicistronic mrna that contains sequences for vpu at the 5' end. c. Required for proper maturation of virus particles d. Prevents transport of CD4 to plasma membrane 3. Vpr: a small regulatory protein found in the virion a. Causes G2 arrest; facilitates nuclear entry of preintegration complex 4. Nef: Produced from multiply spliced early transcripts a. pleiotropic effects- can increase or decrease virus replication b. reduces expression of MHC Class I and CD4 c. enhances virion infectivity d. immunization of macaques with a nef-deficient SIV virus did not prevent their offspring from developing AIDS-like disease Spring

28 POL Proteins Spring

29 Spring 2002 RT 435

30 Integrase Spring

31 Assembly VIII. Assembly of the virus capsid a. Review: structural proteins are translated by free cytoplasmic ribosomes as a polyprotein precursor. b. Frameshifting is used to express replication proteins (RT and INT) c. The capsid precursor protein and the capsid replicative precursor proteins coassemble at the inner surface of the cell membrane. 1. These proteins insert themselves into the cellular membrane via a myristic acid fatty acid which is attached to the amino terminus of the capsid precursor protein. d. The capsid protein precursor contains two copies of a conserved cysteinehistidine box which binds to the viral RNA near its 5' -end 1. The site is referred to as ' Psi' sequence (packaging sequence) e. The complex of capsid protein, replicative enzyme precursor, and viral RNA assemble into a closed spherical virus particle f. Budding through the cell membrane occurs g. Late maturation events include activation of the viral protease 1. This results in the cleavage of the capsid proteins and capsidreplicative enzyme precursor 2. Cleavage results in reorientation of the capsid proteins a. The amino terminus of the protein remains in close contact to the inner surface of the envelope lipid bilayer b. The carboxy terminal proteins assemble into the cone shape core Spring

32 Spring

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES 1 of 7 I. Viral Origin. A. Retrovirus - animal lentiviruses. HIV - BASIC PROPERTIES 1. HIV is a member of the Retrovirus family and more specifically it is a member of the Lentivirus genus of this family.