STUDENT S GUIDE Case Study The origins and evolution of HIV Version 1.2 Anne Fischer Formerly of the Max Planck Institute for Evolutionary Anthropology, Leipzig Dean Madden [Ed.] NCBE, University of Reading
Introduction Prevalence of HIV HIV (Human Immunodeficiency Virus) is the virus that can lead to AIDS (Acquired Immune Deficiency Syndrome) in humans. AIDS is a disease in which the immune system begins to fail, enabling other infections to threaten the lives of patients. Since 1981, when it first began to spread widely, HIV has caused the deaths of 25 million people worldwide. PHOTO BY: Chris Jackson, Getty Images According to current United Nations estimates, HIV will infect 90 million people in Africa, leaving at least 18 million orphaned children there. There are several different forms of HIV: evidence suggests that they originated in Africa, but how are the different forms related to one another and how did they enter the human population? This Case Study uses genetic sequence data from different types of HIV and compares them with SIVs (simian immunodeficiency viruses), which are found in wild chimpanzees and gorillas in Africa. A boy sorts maize at the Reitutsire orphanage in Maseru, Lesotho. The orphanage is supported by Prince Harry s charity, Sentebale. Many adults in Lesotho have been killed by AIDS leaving a generation of over 380,000 orphans to fend for themselves. IMAGE FROM: UN AIDS Global report, July 2008 Question Estimated prevalence of HIV among young adults (aged 15 49) by country in 2008. a. Study the map above. Describe the distribution of HIV. Which countries have the greatest adult prevalence of HIV/AIDS? (Note: the map shows the incidence of HIV/AIDS, not AIDS-related deaths.) 2
Transmission of HIV HIV, the virus that causes AIDS, can be transmitted through: unprotected sex (in semen or vaginal fluid); blood (contact with contaminated material such as needles and contaminated blood transfusions); transmission from mother to child during pregnancy or at birth; breast milk. PHOTO BY: Pascal Le Segretain, Getty Images The virus was characterised in 1983 by a team from the Institut Pasteur in France led by Luc Montagnier. In 2008, Montagnier and a colleague, Françoise Barré-Sinoussi, were awarded the Nobel Prize in Physiology or Medicine for their discovery of HIV. An HIV particle (called a virion) is about 100 nm in diameter. This is about 1/20th of the length of a E. coli cell, and about 1/70th of the diameter of the white blood cells that the virus infects. Luc Montagnier, the co-discoverer of HIV, photographed in 2008. IMAGE FROM: Medical Art Service, Munich / Wellcome Images The basic structure of the Human Immunodeficiency Virus (HIV). The virus s spherical bilipid membrane (yellow) is studded with 72 glycoproteins (green), made from the proteins gp 120 and gp 41. Beneath the membrane, a shell made from the protein p 17 (pink) surrounds the conical core or capsid (yellow) made from p 24 protein. The core contains two identical single strands of RNA (ribonucleic acid). A retrovirus HIV has nine genes, compared to about 25 000 genes in its human host. These include sequences encoding three enzymes required for HIV replication: reverse transcriptase, protease and integrase (encoded by the pol gene). Like all viruses, HIV cannot reproduce by itself. To make new copies of themselves, viruses must infect the cells of living organisms. HIV can only replicate within human cells. HIV is a retrovirus. Retroviruses have RNA (not DNA) as their genetic material. They use an enzyme called reverse transcriptase to reverse-transcribe their RNA into DNA, which can then be integrated into the host s genome and replicated. 3
New virus leaves cell New virus assembled DRAWING BY: Dean Madden, NCBE HIV attaches to CD4 receptors on a T-cell then fuses with the host cell membrane Viral RNA transcribed from DNA Viral RNA (two copies) and enzymes enter the cell Viral protease is needed to process the three viral proteins DNA is transcribed from viral RNA Double-stranded DNA is produced Viral integrase DNA integrates with the host chromosome HIV s replication cycle HIV is replicated in the human host s cells as follows: the virus binds to a protein called CD4 on the surface of the host s immune cells (e.g., lymphocytes). This allows the viral membrane to fuse with the cell membrane, after which it releases the contents of the HIV particle (virion) into the cell; the viral RNA and three enzymes it encodes pass into the hosts cells. The enzymes are a protease, a reverse transcriptase and an integrase, all of which are needed for replication of the virus; the viral RNA is reverse transcribed into DNA; the viral DNA is then integrated into the genome of the host cell by the integrase; the DNA is transcribed back into RNA and translated into proteins that form new viruses. Using this mechanism, up to ten billion new viruses can be produced every day. This rapid replication, coupled with a high mutation rate, contributes to HIV s variability and evolutionary success. 4
HIV is placed within a subgroup of the retroviruses, called lentiviruses (lenti is Latin for slow, and lentiviruses have a long incubation period). This diagram shows the relationship between various lentiviruses. Notice that there are two main forms of HIV: HIV-1 and HIV-2. Other lentiviruses include SIV (simian immunodeficiency virus), BIV (bovine immunodeficiency) virus and FIV (feline immunodeficiency virus) which infect apes and monkeys, cows and cats, respectively. Two types of HIV Two types of HIV infect humans: HIV-1 and HIV-2. HIV-1 is easilytransmitted. It is virulent and is the cause of the majority of HIV infections globally. HIV-1 can be divided into three subgroups: HIV-1-M, HIV-1-N and HIV-1-O, of which HIV-1-M is the most prevalent and has spread around the world. HIV-2 is less virulent than HIV-1 and is not transmitted as easily. It is largely confined to West Africa. The effects of both viruses on humans are similar: HIV-1 and HIV-2 are therefore distinguished by their genomes. HIV denialism Some people, including scientists who are not experts on HIV, have suggested that HIV is not the cause of AIDS. They therefore question the validity of HIV testing and treatment for AIDS. The mainstream scientific community has rejected these claims. Unfortunately, some governments, particularly those in South Africa, have until very recently supported AIDS denialism and encouraged the use of ineffective treatments such as vitamin supplements. This has contributed to the failure of South Africa s response to its AIDS epidemic, although the situation is improving now. Question b. Now you know something about HIV, look once more at the map on page 2. Suggest several different explanations for the distribution of HIV/AIDS shown on the map. 5
Where did HIV originate? Since HIV was discovered in early 1980s, there has been considerable speculation about its origin. One hypothesis suggests that HIV was transmitted to humans from other primates. The close genetic relationship between humans and primates makes it likely that viruses could be transmitted between these species. Non-human primates (e.g., monkeys and apes) carry HIV-like viruses, called SIVs (simian immunodeficiency viruses). Unlike HIV, the viruses that non-human primates carry rarely cause any disease in their hosts. These animals are called asymptomatic carriers (which means that they display no disease symptoms). Studying the evolutionary relationships between strains of HIV and related SIVs from African primates has made it possible to discover more about the likely origin of HIV. By comparing the genetic sequences of HIV and HIVlike viruses from non-human primates, one can identify which species is most likely to have transmitted the virus to humans. Clues from genes and geography Scientists were able to show in 1989 that the RNA sequence of SIV from both captive and wild Sooty mangabeys was very similar to that of HIV-2. This supports the idea that HIV-2 originated in non-human primates. Other evidence supporting the idea of cross-species transmission is the overlapping geographical distribution in west Africa of SIV-infected Sooty mangabeys and humans infected with HIV-2. PHOTO BY: Irwin Bernstein, University of Georgia. Sooty mangabeys are West African primates that carry an SIV which is thought to be the origin of HIV-2. Historical range of the sooty mangabey in West Africa (shown in green). The geographical range of these monkeys corresponds closely with the occurance of HIV-2 in humans. From: Santiago, M. L. et al. (2005) Journal of Virology 79 (19) 12515 12527. 6
The origin of HIV-1 The origin of HIV-1 was unclear for many years. Chimpanzees and gorillas are a potential source, but they are endangered species and it is not easy to get blood samples from living animals in the wild. A breakthrough came when Beatrice Hahn and her colleagues in Brimingham, Alabama, developed a method of isolating DNA and RNA from faecal samples collected from the forest floor. Analysis of faeces from wild chimpanzees and gorillas has revealed the presence of SIV in these species. Comparisons of chimpanzee and gorilla SIV and human HIV-1 sequences were made by a team led by Paul Sharp at the University of Nottingham, in co-operation with Hahn and many other researchers. The work showed that these viruses are very similar. The natural habitat of chimpanzees and gorillas coincides with the epicentres of HIV-1 epidemics. Furthermore, the central African region encompassing Gabon, Cameroon, Equatorial Guinea and the Republic of Congo, is the only place where all three subgroups of HIV-1 (M, N and O) are found. Sequences of SIVcpz (the SIV that infects chimpanzees) and SIVgor (the SIV that infects gorillas) that resemble HIV-1 sequences most closely have been found in chimpanzees and gorillas inhabiting the same geographical region. How could transmission between species occur? Chimpanzees and gorillas are the closest living relatives of humans. These species, as other primate species, are commonly hunted for food (bushmeat) and orphan chimps are sometimes kept as pets. Such pets are a natural reservoir for the disease and they could transmit SIVcpz to humans. With extensive logging of tropical rainforest, access to previously remote areas is now possible, which further sustains the bushmeat trade. PHOTO BY: Thomas Lersch, Wikimedia Commons PHOTO BY: Mila Zinkova, Wikimedia Commons Chimpanzee. Male silverback gorilla. 7
Sequence analysis The aim of this exercise is to study the similarities between SIV and HIV-1 sequences. This will allow you to investigate the potential transmission of these viruses between great apes and humans. The data provided are 16 nucleotide sequences from the pol gene of the HIV and SIV viruses from chimpanzee, gorilla and human. There are six human sequences, two from each of the three HIV subgroups (M, N and O), two gorilla sequences and eight chimpanzee sequences. The data file is called: DNA-HIV1andSIV.geneious The analysis will be performed using a programme called Geneious. This software can align sequences and build phylogenetic trees. The nucleotide sequences will first be translated into protein sequences, which will then be aligned. From the alignment of the protein sequences, you will build a phylogenetic tree. This will show which sequences are more closely related to one another. 1. Double click on the document named DNA-HIV1andSIV. geneious. This will start the Geneious software and load the file of genetic sequence data into the programme. Hint: if a box appears over the Geneious start-up screen, saying that your trial of the Pro version has ended, click on Use Geneious Basic. 2. The 16 DNA sequences will now open in Geneious: The names of the sequences are shown here. The DNA sequences are in this central window. 8
3. You can use the magnifying glass buttons to zoom in on the nucleotide data: Zoom buttons Questions c. Use the magnifying glass buttons to zoom in on the sequence data. How can you tell that it is DNA sequence data and not RNA sequence data? d. What sort of genetic information does HIV (a retrovirus) have? e. How has the data therefore been processed before it was given to you? 4. Select all 16 sequences at the same time, by clicking on the file name in the top window so that it is highlighted: 9
5. Click on the Translate button to convert the DNA sequence data into protein sequence data: Translate button 6. A box will appear, asking you to choose a version of the genetic code to use. Look at the options available, then choose Standard and click OK. Note Although the genetic code is often said to be universal the same in all living things this is not quite true. There are some minor variations in different groups of organisms. Hence this dialogue box, which allows you to choose which version of the code you wish to use. A new file will appear in the top window, containing 16 protein sequences derived from the original DNA sequences: 10
7. Use the magnifying glass button again to zoom in on the sequence data and check that the sequences are in fact proteins made of amino acids (the single-letter amino acid codes are used here): Asp D Aspartic acid Ala A Alanine Glu E Glutamic acid Gly G Glycine Arg R Arginine Val V Valine Lys K Lysine Leu L Leucine His H Histidine Ile I Isoleucine Asn N Asparagine Pro P Proline Gln Q Glutamine Phe F Phenylalanine Ser S Serine Met M Methionine Thr T Threonine Trp W Tryptophan Tyr Y Tyrosine Cys C Cysteine Amino acid codes The three-letter and single letter codes for the 20 amino acids that are found in proteins. Geneious uses the single-letter codes to show the different amino acids. 8. The protein sequences should already be aligned, but before creating a phylogeny, you will need to ensure that they are. Select the protein sequences in the top window, then click the Alignment button: Alignment button 11
9. A box will appear, asking you to choose a method of alignment. Only one method is possible with the basic Geneious software, so select Geneious Alignment then click OK. The alignment will take a few minutes to complete (slightly longer on a slow computer): Tree button 10. Select the aligned protein sequences ( Alignment of 16 sequences ) in the top window, then click on the Tree button to create a phylogeny. 12
A box will appear, offering some options for the tree building. Select the values shown below and click OK. Select Jukes- Cantor and Neighbour- Joining here. Technical note The Jukes-Cantor distance model assumes that all amino acid substitutions (mutations) happen at the same rate (1 in 20 or 5%). Other mathematical models assume that different amino acids mutate at different rates. The Neighbor-Joining method is a quick and popular mathematical model for calculating genetic distances and drawing trees. Other methods will produce slightly different results (and take longer to do it). 11. A tree will be produced in the lower central window. Re-size the other windows so that you can study the tree. The software will cluster similar sequences closer together. You now have a phylogenetic tree of sequences from the three subgroups (M, N and O) of the HIV-1 family and their relationship to SIV sequences from chimpanzees and gorilla. 13
the origins and evolution of hiv Questions f. Mark, on a paper print out of the tree, using three different colours or symbols, the branches of the tree that derive from gorilla, chimpanzee and human viruses. g. Describe the locations of the HIV sequences in the tree (for example, do they form any clusters or groups, or are they scattered throughout the branches of the tree?) h. Do the HIVs appear to be more closely related to each other, or to some of the SIV sequences? i. Do you think that HIV-1 could have originated more than once, and if so, what was the source on each occasion? j. Does the geographical distribution of SIV-infected apes overlap with areas of HIV-1 epidemics? Compare the map on page 2 with the one below and, if you have access to the internet: www.aidsinafrica.net/ map.php and www.unaids.org/en/ Further reading Avert, a UK-based AIDS charity, has a website with comprehensive and authoritative information about HIV/AIDS: www.avert.org 14
A phylogenetic tree of sequences from the three subgroups (M, N and O) of the HIV-1 family and their relationship to SIV sequences from chimpanzees and gorilla. The codes after the virus names refer to sampling areas. TAN = Tanzania; CAM = Cameroon; GAB = Gabon. 15