Biology/CS 123A Bioinformatics I

Bioinformatics SIX Retroviruses: HIV Sami Khuri Department of Computer Science San José State University Biology/CS 123A Fall 2012

Retroviruses: HIV








Intracellular Parasites gag, pol, & env genes HIV-1 Immune System Macrophage T-Cells AIDS

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What is a Virus? Viruses: Small living particles that can infect cells and change how the cells function. Infection with a virus can cause a person to develop symptoms. The disease and symptoms that are caused depend on the type of virus and the type of cells that are infected. www.medterms.com ©2010 Sami Khuri

Ancient Egypt and Viruses

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Some Terminology (I) • Virion: A complete virus particle with its DNA or RNA core and protein coat as it exists outside the cell. Also called a viral particle. • Capsid: the outer covering of protein surrounding the nucleic acid of a virus. ©2010 Sami Khuri

Some Terminology (II) • Pathogen: patho: disease, gen: producer A disease producer. The term pathogen is commonly used to refer to infectious organisms including: bacteria, fungi, and viruses, such as HIV. • Virulence: the ability of an infectious agent to produce disease. Many viruses are virulent sometimes and asymptomatic at other times. ©2010 Sami Khuri

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Some Terminology (III) Immunodeficiency: The result when the immune system fails to protect the host from disease-causing agents or from malignant cells. Acquired immunodeficiency: is the loss of immune function and results from exposure to various agents. It is acquired because the genetic or developmental deficiency in the immune system was not present at birth. ©2011 Sami Khuri

HIV Case Study • Why have promising AIDS treatments, like drug azidothymidine (AZT) proven ineffective in the long run? • Why does HIV kill people? • Why are some people resistant to becoming infected or to progressing to disease once they are infected? • Where did HIV come from? ©2011 Sami Khuri

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Retrovirus • A retrovirus is a single-stranded RNA virus that employs a double-stranded DNA (dsDNA) intermediate for replication. • The RNA is copied into DNA by the enzyme reverse transcriptase. • The dsDNA is integrated into the host chromosomes, from which it is transcribed to produce the viral genome and proteins that form new viral particles. ©2010 Sami Khuri

Complimentary DNA cDNA A,C,G,T

Adenine Guanine Cytosine Thymine

(A) (G) (C) (T)

A,C,G,U

Adenine (A) Guanine (G) Cytosine(C) Uracil (U)

20 Amino Acids

{A-Y}-{BJOUX}

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Provirus Formation

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Transcription of Viral Genes

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HIV • The human immunodeficiency virus (HIV) is the virus that causes acquired immune deficiency syndrome (AIDS). • HIV moves from person to person when a bodily fluid containing the virus, usually blood or semen, carries the virus from an infected person directly onto a mucous membrane (membrane lining all body passages that communicate with the air) or into the bloodstream of an uninfected person. ©2011 Sami Khuri

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What is HIV? • Like all viruses, HIV is an intracellular parasite. • It is incapable of an independent life and is highly specific in the cell types it afflicts. • HIV parasitizes components of the human immune system: macrophages and T cells. • HIV uses the enzymatic machinery and energy found in these cells to make copies of itself, killing the host cells in the process. http://www.niaid.nih.gov/factsheets/howhiv.htm ©2011 Sami Khuri

HIV Transmission • The HIV virus can be transmitted during heterosexual sex, homosexual sex, oral sex, needle sharing, transfusion with contaminated blood products, childbirth, and breastfeeding. • The HIV virus has spread via different routes in different regions since first recognized by medical professionals in 1981. ©2011 Sami Khuri

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Macrophages and T Cells • Macrophage - a large immune system cell that devours invading pathogens and other intruders. Stimulates other immune system cells by presenting them with small pieces of the invaders. • CD4+ T cells - white blood cells that orchestrate the immune response, signaling other cells in the immune system to perform their special functions. Also known as T helper cells, these cells are killed or disabled during HIV infection. ©2011 Sami Khuri

HIV is a Lentivirus • HIV is a retrovirus that belongs to the class of lentiviruses: – Lentiviruses are slow viruses. The course of infection with these viruses is characterized by a long interval between initial infection and the onset of serious symptoms. • Other lentiviruses infect nonhuman species. – Example • Feline immunodeficiency virus (FIV) infects cats • Simian immunodeficiency virus (SIV) infects monkeys and other nonhuman primates. ©2011 Sami Khuri

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FIV and SIV • Like HIV in humans, these animal viruses primarily infect immune system cells, often causing immunodeficiency and AIDS-like symptoms. These viruses and their hosts have provided researchers with useful, albeit imperfect, models of the HIV disease process in people. • Humans inherited HIV from chimpanzees and mangabeys and since then, HIV has diversified and continues to do so. ©2011 Sami Khuri

How Does HIV Cause AIDS? • The human body responds to HIV infection by destroying virions floating in the bloodstream and by killing its own infected cells before new virions are assembled and released. • Ultimately, the supply of CD4 helper T cells depletes and the immune system collapses. ©2011 Sami Khuri

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Disease Progression

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Stages of HIV Infection

Stages of HIV infection in a typical untreated patient ©2011 Sami Khuri

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Geographic Distribution of HIV

30–36 million people living with HIV, 2007 ©2011 Sami Khuri

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HIV-1 Virion Each component of env consists of: -a cap made of three gp120, and - a stem consisting of three gp41 molecules that anchor the structure in the viral envelope

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Gag Pol and Env Retroviruses encode at least three proteins that take in viral replication: the products of the gag, pol, and env genes. – gag encodes for the core proteins, structural virion components. It plays a role in the maturation of the RNA genome. – pol encodes the reverse transcriptase and the integrase – env encodes the structural protein that surrounds the virus. This protein is necessary for the virus to leave the cell to infect other cells. ©2011 Sami Khuri

Genome of HIV-1

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HIV Genomic Organization

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Difficulty of HIV Treatment • HIV uses the host cell’s own enzymatic machinery: – its polymerase – its ribosome – its tRNAs • Drugs that interrupt the life cycle of the virus are almost certain to interfere with the host’s cell enzymatic as well, thus causing serious side effects. ©2011 Sami Khuri

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Inhibit fusion

Potential Drugs Inhibit integrase

Inhibit RT

Inhibit protease

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Azidothymidine (AZT) Blocks Reverse Transcriptase

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Azidothymidine Results • AZT worked in early tests: – Effectively halted the loss of macrophages and T cells in AIDS patients. • AZT can cause serious side effects because it sometimes fools DNA polymerase and interrupts DNA synthesis in host cells. • After a few years of use, patients stop responding to treatment. ©2011 Sami Khuri

Mutations in Reverse Transcriptase

Over time, the active site in the reverse transcriptase changes, making it less likely to mistake AZT for the normal nucleotide. ©2011 Sami Khuri

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How Does the Change Occur? • The reverse transcriptase is error prone and the HIV genome does not have error-correcting enzymes – Over half of the DNA transcripts produced by the reverse transcriptase contain at least one mistake in their nucleotide sequence. • HIV has the highest mutation rate of any virus or organism observed to date: – Thousands of generations of HIV replications take place within each patient during the course of infection and a single strain of HIV can produce hundreds of different reverse transcriptase variants over time. ©2011 Sami Khuri

Natural Selection • One or more of the variants will contain an amino acid substitution that lessens reverse transcriptase affinity for AZT. • If the patient takes AZT, the replication of wild-type HIV variants is suppressed but the resistant mutants will still be able to synthesize some DNA and produce new virions. • This is an example of evolution by natural selection. ©2011 Sami Khuri

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HAART

• Current treatments use combinations of drugs (treatment cocktails): – E.g. two reverse transcriptase inhibitors plus a protease inhibitor.

• Multidrug treatments are known as Highly Active AntiRetroviral Therapy or HAART. ©2011 Sami Khuri

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Number of People Receiving Antiretroviral Drugs Millions

3.0 2.8

North Africa and the Middle East

2.6 2.4

Eastern Europe and Central Asia

2.2 2.0

East, South and South-East Asia

1.8

Latin America and the Caribbean

1.6 1.4

Sub-Saharan Africa

1.2 1.0 0.8 0.6 0.4 0.2 0.0

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end2007 Data provided by UNAIDS & WHO, 2008.

Some People are Resistant to HIV • In the early 1990s, work from several laboratories demonstrated that some people remain uninfected even after repeated exposure to the virus and some people who are infected with the virus survive many years longer than expected. • Resistant individuals have unusual forms of the coreceptor molecules and these mutant proteins thwart HIV entry. ©2011 Sami Khuri

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Human CC-CKR-5 (CCR5) – CCR5 is a cell surface protein found on white blood cells. – The CCR5 function is to bind chemokines, which are molecules released as signals by other immune system cells. • When a white blood cell is simulated by chemokines binding to its receptors, the cell moves into inflamed tissues to help fight an infection. – CCR5 is also exploited as a coreceptor by most sexually transmitted strains of HIV-1 ©2011 Sami Khuri

CCR5 Function in HIV-1 Infection

coreceptor CD4

HIV entry into the cell requires binding to a CD4 molecule and, in the majority of cases, to a coreceptor, either chemokine coreceptor 4 (CXCR4) or 5 (CCR5).

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Rong Liu et al. • A CKR-5 allele (CCR5 allele) present in the human population appears to protect homozygous individuals from sexual transmission of HIV-1 strain R5. • These individuals appear to have inherited a defective CKR-5 allele that contains an internal 32 base pair deletion. • The deletion occurs within the coding region and results in a frame shift. • The encoded protein is severely truncated and cannot be detected at the cell surface.

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Amino acids as the result of 32bp deletion

10bp repeat

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Genotyping Individuals • Samson et al. took DNA samples from a large number of individuals from different parts of the world, examined the gene for CCR5 in each individual and calculated the frequency of the normal and ∆32 alleles in each population. ©2011 Sami Khuri

CCR5-∆32 Allele Distribution • Gene frequency of about 10% was observed for CCR5-∆32 in populations of European descent. • As we move away from northern Europe, both to the east and to the south, the frequency of the ∆32 allele declines. • Outside of Europe, in the Middle East, and western Asia, the ∆32 allele is virtually absent. ©2011 Sami Khuri

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www.hivgene.com ©2011 Sami Khuri

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Genetic Drift Hypothesis • The ∆32 allele first appeared and achieved a high frequency among the Vikings and then was disseminated across Europe during the Vikings raids of the 8th, 9th, and 10th centuries.

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Coreceptor Antagonists • Molecular biologists are trying to design drugs that mimic the effect of the resistance alleles. • One approach is to find small molecules that bind to the CCR5 protein on the surface of host cells and block HIV’s attempt to use the protein as coreceptor: – Maraviroc (an entry inhibitor) is the first CCR5 coreceptor antagonist to receive marketing approval from the Food and Drug Administration (FDA) for the treatment of CCR5-tropic human immunodeficiency virus (HIV) infection as part of an optimized antiretroviral regimen in treatmentexperienced patients. ©2011 Sami Khuri

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Different Strains of HIV • Relatively benign strains of HIV are known to exist. – Sydney Bloodbank Cohort: • 8 transfusion patients with HIV transmitted from a blood donor have no symptoms or very slow decline in CD4 T cell counts. • HIV uses CXCR4 as the coreceptor instead of CCR5. • HIV genome lacks gene for a viral protein called Nef which helps HIV virions gain entry into host cell, boosts viral replication and helps HIV-infected cells evade host’s immune system.

• These strains of HIV may be transmitted from host to host at low rates. ©2011 Sami Khuri

HIV-1 and HIV-2 • HIV-1 is more common worldwide than HIV-2. • HIV-1 has been discussed so far. • HIV-2 is a related virus that humans acquired from a different primate species. • HIV-2 is less damaging to its hosts than HIV-1: – Individuals infected with HIV-2 progress to AIDS but at a slower rate. YouTube: HIV Replication 3D Medical Animation and www.hivgene.com/what-is-ccr5.php ©2011 Sami Khuri

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The Origin of HIV • Hahn et al. (2000) sequenced the gene that codes for reverse transcriptase in several SIVs and compared them to sequences found in a variety of HIV strains. • The data were used to estimate the relationships among these viruses (phylogenetic tree). • The two main types of HIV, HIV-2 and HIV-1, were transmitted to humans from different sources: – HIV-2 originated in sooty mangabeys, while HIV-1 was originally transmitted to humans from chimpanzees. – HIV-2 is prevalent in West Africa. ©2011 Sami Khuri

The Origin of HIV Virus

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Refinement of the phylogenetic tree based on the sequences from the gene that codes for viral surface proteins

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HIV-1 Subgroups • HIV-1 strains cluster into three distinct groups, called M, N, and O. – Subgroup M is responsible for the bulk of the global AIDS epidemic.

• Each subgroup is closely related to a different strain of SIVcpz.. • The phylogenetic tree provides evidence that HIV-1 was transmitted to humans from chimps on at least three different occasions. ©2011 Sami Khuri

Medical Lessons from Phylogeny • Chimpanzees are model organisms: – Important questions yet to be answered include: • How common is SIVcpz in the wild? • How is it transmitted? • Why does SIVcpz not make chimps ill?

• The tree is a clue as to why it has been so hard to develop an effective vaccine: – Many subgroups of HIV-1 exist. – Within a single subtype and in a single infected person, the virus also changes constantly, so a vaccine will need to protect against multiple subtypes. – Transmissions from chimps to humans happened repeatedly in the past and will likely continue in the future. ©2011 Sami Khuri

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