Going Wild: Lessons from Naturally Occurring T-Lymphotropic Lentiviruses. VandeWoude & Apretrei Clinical Microbiology Reviews 27:728 (2014)

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August Long
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1 In ancient times, citizens sought answers to their most important inquiries by consulting oracles. Myths were part of daily life, and mythological creatures were feared or defeated. Oedipus deciphered the riddle of the Sphinx, a creature that was part primate (human) and part feline (lion). In modern times, many astute scientists have worked diligently to decipher a modern riddle, i.e., understanding human immunodeficiency virus (HIV) and using this knowledge to halt the AIDS pandemic. Although we endeavor to solve this riddle with the tools of science, we should keep in mind that, like Oedipus, we may find clues hidden in the intricate biology of primate and feline lentiviruses. Going Wild: Lessons from Naturally Occurring T-Lymphotropic Lentiviruses. VandeWoude & Apretrei Clinical Microbiology Reviews 27:728 (2014)

2 Retroviruses Subfamily: Spumaretrovirinae (1 Genus) Subfamily: Orthoretrovirinae (6 Genera) Genus: Alpharetrovirus (9 Species) Genus: Betaretrovirus (5 Species) Genus: Deltaretrovirus (4 Species) Genus: Epsilonretrovirus (3 Species) Genus: Gammaretrovirus (17 Species) Genus: Lentivirus (9 Species) Len-viruses Species: Bovine immunodeficiency virus Species: Caprine arthritis encephalitis virus Species: Equine infectious anemia virus Species: Feline immunodeficiency virus Species: Human immunodeficiency virus 1 Species: Human immunodeficiency virus 2 Species: Puma lentivirus Species: Simian immunodeficiency virus Species: Visna/maedi virus Over 40 nonhuman primate (NHP) species harbor species- specific simian immunodeficiency viruses (SIVs). More than 20 species of nondomesdc felids and African canids demonstrate seroreacdvity against feline immunodeficiency virus (FIV) andgens. Rarely detected increased morbidity or impaired fecundity/survival of naturally infected SIV- or FIV- seroposidve versus - seronegadve animals Cross- species transmissions of these agents are rare in nature lendviral infecdons have been idendfied in horses, goats, sheep, and camle, the majority of these infecdons appear to be clinically silent Sheep and Goat lendviral infecdons may result in neurological disorders, arthrids, and pneumonia, while horse lendviral infecdons result in recurrent fever and blood diseases

3 HIV-1 Entry HIV, and other primate lentiviruses, infects CD4 + T lymphocytes and will cause them to die during a productive infection. HIV infects other cells but does not cause cell death, at least at the same rate as CD4 T cells: Natural killer cells CD8+ killer T-cells Macrophages Cell of the nervous system (e.g., astrocytes, neurons, glial cells, and brain macrophages) Dendritic cells

4 HIV Entry The infectious virus is always specific for CD4 and CCR5 present on memory T cells and macrophages The rapid progression to AIDS can be associated with a switch in co-receptor preference to CXCR4 HIV enters cells via a cellular receptor and co-receptor Major cellular receptor: CD4 present on T-lymphocytes (also present in low concentrations on macrophages) Co-receptor on T lymphocytes: CCR5 (memory) or CXCR4 (naïve) Co-receptor on macrophages: CCR5

Schematic of HIV Virion Different HIV particles have highly variable protein spikes, Glycoprotein 120 or gp120 and random mutations in the HIV genome causes

5 Schematic of HIV Virion Different HIV particles have highly variable protein spikes, Glycoprotein 120 or gp120 and random mutations in the HIV genome causes random changes in the structure of the spikes on different HIV virion. Extends out from the viral envelope as a trimer. The CD4 molecule interacts with gp120

6 CD4 s role in antigen recognition The structure of CD4

to be exposed The gp41 molecule darts out and pierces the cell membrane of the macrophage or

7 GP120 trimers bind with CD4 and change shape The union between HIV and its host T-lymphocyte gp120 interacts with CCR5 or CXCR4 (fusins), causing further shape changes and causes the viral gp41 trimers to be exposed The gp41 molecule darts out and pierces the cell membrane of the macrophage or T-lymphocyte. CD4 Viral envelope and cell membrane lipids fuse, delivering the HIV nucleocapsid into the cytoplasm

HIV membrane fusion The infecdon with HIV1 isinidated by the interacdon of viral gp120 envelope protein with CD4 protein expressed mainly by cells of the T lymphocyte and macrophage lineages.

8 HIV membrane fusion The infecdon with HIV1 isinidated by the interacdon of viral gp120 envelope protein with CD4 protein expressed mainly by cells of the T lymphocyte and macrophage lineages. The interacdon between gp120 and CD4 induces a conformadonal change in gp41, resuldng in the inserdon of the N- terminal hydrophobic fusion- pepdde region into cell membrane. Further intra- protein interacdon between the N- and C- terminal of the gp41 ectodomain regulates the membrane fusion and the entry of HIV contents into cytoplasm. Besides the CD4 receptor, coreceptors CCR5 or CXXR4 also help the entry of HIV. The binding of gp120 to CD4 and its affinity for coreceptor largely depend on the sequence pamern of its V3 and V4 variable regions.

9 The replication cycle of HIV 1 and 2 Uncoating Removal of the genome from the nucleocapsid. Cellular enzymes strip the protein capsid away and the genome is released. Capsid removal When the capsid is removed, RNA, two proteins (p24 and p17), and reverse transcriptase are released Synthesis of viral nucleic acid and protein ssrna is reverse transcribed into a dsdna strand, which migrates to the nucleus of the host cell The DNA molecule is incorporated into the cell s DNA at a random site by integrase, and becomes part of the cell s 46 chromosomes. The viral DNA molecule within the chromosome is termed a provirus. This phenomenon is known as lysogeny

10 Assembly The provirus DNA is transcribed into RNA. Some is mrna that is translated into viral proteins (e.g., gp120, reverse transcriptase, etc.). Other full-length RNA strands are used to construct a new generation of HIV genomes. Genomes, capsid proteins, and enzyme molecules are assembled at the edge of the cell, forming a circular structure that binds to the cell membrane The replication cycle of HIV 1 and 2 Protease snips out enzyme molecules and capsid proteins, then sections the capsid proteins into segments that unite and form an icosahedral capsid. Collapses to yield a conical capsid surrounding the RNA genome and enzymes. Note that p120 and gp41 extend from the cell s membrane as spikes. The assembled viral nucleocapsid localizes to just under the membrane of the cells and buds through the membrane to form the envelope

11 The replication cycle of HIV 1 and 2 Budding Virus coats itself with the cell membrane and pinches off, taking the membrane and spike proteins with it as the envelope. The virus is then released to the extracellular environment and HIV replication is complete.

12 The capsid and matrix proteins and HIV enzymes need to be cleaved by a protease inside of HIV for the virus to be infectious. Adapted from Vella, S., et al., AIDS Soc. 4 (1996):

13 Electron Micrograph Showing Mature HIV Particles Courtesy of Louisa Howard, Dartmouth College, Electron Microscope Facility

14 Figure 9-13

15 Basic retroviral genome

16 HIV genome

17 Know what gag, pol, and env encode, pls Important drug targets burst of replication when infected T cells are stimulated necessary for the reverse transcription of RNA to DNA enhances the movement of genetic messages into the cytoplasm

18 One of the normal functions of the nef gene is to downregulate host antigen presentation. It does so by binding to HLA class I receptors via its binding motif thus interfering with intracellular HLA class I antigen processing

19 Figure 9-15 part 1 of 4

20 Figure 9-15 part 2 of 4

21 Figure 9-15 part 3 of 4

22 Figure 9-15 part 4 of 4

23 Primary (acute) HIV infection Fever, pharyngitis, adenopathy, and rash Measures of virus and symptoms decline precipitously with seroconversion Early infection, virus completely homogeneous, late infection, viral heterogeneity Immune response to virus or loss of CD4 memory cells Loss of CD4 T cells and opportunistic infections

24 CD4 counts with time for individual patients (months after onset of primary illness)

25 B T Grenfell et al. Science 2004;303: years!

26 Viral infecdon results in producdon of INTERFERONS rednoic acid inducible gene- 1 (RIG- 1) dsrna- dependent protein kinase (PKR) The host type I interferon response to viral and bacterial infec-ons Andrea K PERRY, Gang CHEN, Dahai ZHENG, Hong TANG and Genhong CHENG, Cell Research

27 Restriction factors = proteins that prevent viral replication, transmission, etc. 1. Proteins that exhibit antiviral activity 2. Virus infection signaled by TLRs & other PRRs, resulting interferon production 3. Constitutively expressed or induced by interferons 4. Often themselves antagonized by virus proteins (virulence factors) 5. Most often, cell-autonomous (act within the cells) 6. Exceptions are the interferons themselves: cytokines that act upon the interferon-producing cell (autocrine) and between cells (paracrine) 7. Cell intrinsic factors can be increased by interferons 8. Restriction factors target specific steps in the HIV-1 life cycle, but unlike antiretroviral drugs, they do so in a way that makes it difficult for HIV-1 to evolve resistance to the inhibitor through simple evasive mutations (maybe) 9. Intrinsic Cellular Defenses Are Determinants of Viral Host Range 10. Blocks to cross-species transmission are imposed by APOBEC3G and TRIM5 proteins that appear particularly powerful a. They are especially potent anti-viral proteins b. Constitutively expressed lentiviral target cells: T cells and macrophages

28 APOBEC-3 APOlipoprotein B Editing Catalytic subunit-like 3 (cytidine deaminases) APOBEC3G (384 amino acids, 46.4 kda) is the prototype antiretroviral cytidine deaminase Expressed in virus infected cells and incorporated into progeny virions Upon virion entry into a new target cell, APOBEC3G acts during reverse transcription, primarily during the synthesis of the negative sense DNA strand Deaminates the C4 position of 2 -deoxycytidine producing 2 -deoxyuridine APOBEC3G preferentially acts on the third cytosine of the sequence 5 -CCCA-3 Causes major disruption of the coding potential of the viral genome Viral counter measures: Vif proteins bind to APOBEC3 and recruit proteosome degradation. SIVcpz Vif and SIVsmm Vif both active against HIV-1 and HIV-2 APOBEC3A, APOBEC3B, APOBEC3D, APOBEC3F, and APOBEC3H,

29 TRIM5α TRIpartite Motif family of proteins Targets the incoming viral capsid prior to reverse transcription Consequence, capsid disruption and degradation Acts to promote proteosome-mediated capsid degradation, but proteosome inhibitors do not block anti-viral activity SPRY domain highly variable in primates Dictates the retroviruses that are restricted by a particular TRIM5 variant Human TRIM5α largely ineffective against primate lentiviruses, but monkey TRIM5α very effective A proposed model of TRIM5a activity suggests that TRIM5a forms a complementary threedimensional lattice around the incoming capsid.

30 Genes encoding APOBEC3G and TRIM5α are under postive selection in human beings They have among the highest dn/ds ratios of all human genes What is an dn/ds ratio? Remember this selection pressure occurred in the distant past, long before HIV infected human beings

31 Does Variation in Restriction Factors Contribute to AIDS Susceptibility in Human Beings? Some variation in HIV/AIDS susceptibility in humans can be attributed to CCR5 (rare) and MHC polymorphisms (e.g., HLA B57) Subpopulations of humans encode inactivating or destabilizing polymorphisms in genes encoding TRIM5α, APOBEC3B, APOBEC3H Variation among APOBEC3H haplotypes in terms of antagonism by Vif. Polymorphic mutations are found in TRIM5a (H43Y) and APOBEC3G (H186R) that decrease activity Actual resistance or sensitivity to HIV correlated with TRIM5 or APOBEC3 polymorphisms in human beings has not been consistently found! Resistance due to polymorphisms in TRIM5 family members in macques HAS been shown How can we use this information: inhibitors of Vif has been identified

32 HIV

34 Rapid tests for HIV antibodies C-control goat anti-human Ig T-HIV1, gp41; HIV2, gp36 Saliva, finger stick, serum, etc applied C-unknown T-unknown Serum or plasma, only 3

Invalid Results FOR A TEST TO BE VALID A CONTROL LINE MUST BE PRESENT AND THE SAMPLE PORT MUST CONTAIN FULL RED COLOUR Uni-Gold Recombigen HIV-1/2 REPORT AS INVALID REPORT AS INVALID REPORT AS

line present Control line present No red color at Sample Port No test line present Control line present Not full red color at Sample Port Test line present Control line present No red color at Sample

The test should be repeated in duplicate with fresh devices.

35 Invalid Results FOR A TEST TO BE VALID A CONTROL LINE MUST BE PRESENT AND THE SAMPLE PORT MUST CONTAIN FULL RED COLOUR Uni-Gold Recombigen HIV-1/2 REPORT AS INVALID REPORT AS INVALID REPORT AS INVALID REPORT AS INVALID REPORT AS INVALID Test line present No control line present Full red color at Sample Port No test line present No control line present Full red color at Sample Port No test line present Control line present No red color at Sample Port No test line present Control line present Not full red color at Sample Port Test line present Control line present No red color at Sample Port No pink/red line appears in the device window adjacent to word "Control" whether or not a pink/red line appears in the device window adjacent to word "Test". The test should be repeated in duplicate with fresh devices. No pink/red line appears in the device window adjacent to word "Control" whether or not a pink/red line appears in the device window adjacent to word "Test". The test should be repeated in duplicate with fresh devices. Red color is not seen in the Sample Port. The test should be repeated in duplicate with fresh devices. Red color is not seen in full sample well. White of sample pad remains. The test should be repeated in duplicate with fresh devices. Red color is not seen in the Sample Port. The test should be repeated in duplicate with fresh devices.

probed with patient serum If the patient has been

36 HIV Western Blot Confirmatory test to exclude false-positive results Nitrocellulose membrane is probed with patient serum If the patient has been exposed to HIV, + bands will be observed on the Western blot

37 Discussion points: Who developed the first commercialized HIV test? What did it measure? What is being measured in an Elisa or in a Western blot assay? Why did it take three or four years to develop an assay? Is the assay perfect in diagnosing HIV infection? Why or Why not? Were there alternatives to measuring antibody specific to HIV in screening the blood supply? Did the blood banking industry try them? Which groups were in favor of screening donors, which groups were against?

39 Biosensors and Bioelectronics Volume

Human Immunodeficiency Virus

Human Immunodeficiency Virus Virion Genome Genes and proteins Viruses and hosts Diseases Distinctive characteristics Viruses and hosts Lentivirus from Latin lentis (slow), for slow progression of disease