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AIDS researchers from Ulm to shed light on the success of the HIV-1 M group

Frank Kirchhoff, AIDS researcher at the University of Ulm, has come up with a plausible explanation for the pandemic spread of the human immunodeficiency virus 1 (HIV-1) M group and its ability to adapt to the human immune system. In a recent study with colleagues from Germany and abroad, Kirchhoff used molecular biology methods to investigate the group and compare it with the relatively rare HIV-1 N group. Kirchhoff hopes that the molecular differences will provide his team with insights into the evolutionary adaptation of HIV-1 group M viruses.

Kirchhoff’s current and previous investigations are based on the assumption that the different geographical distribution of the four HIV-1 groups (M, N, O, P) is the result of the different adaptation of the virus to its human host. The HIVs originated in chimpanzees and gorillas and were subsequently transferred to humans. In 1940, HIV-1 M was transferred from a chimpanzee to humans. It is by far the most common type of HIV and the principal cause of the AIDS pandemic, accounting for around 60 million infected people. Around a dozen people have been infected with HIV-1 N, which is also transmitted by chimpanzees. With one exception, all HIV-1 N infected people live in Cameroon. The O and P groups are phylogenetically more closely related to the viruses found in gorillas. HIV-1 O has led to the infection of tens of thousands of people in Cameroon and neighbouring countries, while the P group virus has so far only been detected in two people in Cameroon.

HIV-1 M group viruses have a fully functional Vpu protein

Dr. Daniel Sauter, first author of the paper published in PLOS Pathogens. © University of Ulm

Pandemic HIV-1 group M viruses have developed a fully functional Vpu protein. This viral protein is a specific antagonist of the human restriction factor tetherin (BST-2) and can also degrade the primary human CD4 receptor. Vpu, which is a small viral transmembrane protein 80 to 82 amino acids long, most likely has additional functions. “This is typical for retroviruses such as HIV. They are very variable and are able to develop multitasking tools to degrade or modify anything in the host cell that prevents their replication,” said Kirchhoff. However, tetherin and CD4 appear to be the most important Vpu antagonists. The protein domains that switch off human virus defence mechanisms are known in detail. “It has been shown that specific amino acid residues in the transmembrane of human tetherin are critical for interacting with Vpu.”

The situation in HIV-1 group N viruses is somewhat odd as the viruses have developed modest anti-tetherin activity, but concomitantly lost the ability to degrade CD4. This was the starting point for the researchers from Ulm and the reason that they decided to take a detailed look at the molecular differences between HIV-1 N and M strains. 

In the aforementioned study, which was published in the journal PLOS Pathogens, Kirchhoff’s team analysed the Vpu protein of eight group N viruses. They found that the Vpu proteins of the HIV-1 N group have four amino acid substitutions in their transmembrane domain which allow them to interact efficiently with human tetherin. However, despite these adaptive changes most N group Vpus antagonise human tetherin very poorly and fail to down-modulate other immune system constituents (CD4, NTB-A and CD1d). These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain which disrupt putative adaptor protein binding sites. As a consequence, N group Vpus exhibit aberrant intracellular localisation and/or fail to recruit the ubiquitin ligase complex to induce tetherin degradation. In contrast, HIV-1 M strains have highly conserved sequences in this domain. 

HIV-1 N group counteracts tetherin effectively

Kirchhoff’s team had almost completed the study and clarified the reasons for the functional differences observed between M and N strains when in January 2011 they came across a male patient living in Paris. The 57-year-old patient developed fever, rash and other symptoms about a week after returning from Togo in West Africa and was diagnosed with the first HIV-1 N infection outside of Cameroon where a dozen HIV-1 N infections had previously been diagnosed. The researchers from Ulm therefore decided to take a closer look at the virus and include it in their study.

The investigations came up with an unexpected result: the new HIV-1 N virus had apparently acquired mutations and contained intact cytoplasmic motifs, which enabled it to counteract tetherin as effectively as the Vpus of pandemic HIV-1 M strains. It might just be a single case, but the number of HIV-1 N virus infections could increase in future and needs to be carefully monitored. That aside, the case of the 57-year-old patient suggests that HIV-1 group N viruses are still adapting to their potential hosts, i.e. humans, especially through the modification of the Vpu protein that enables the virus to remove tetherin from the surface of human cells.

Group N viruses have increased transmission fitness potential

“Of course, other HIV-1 strains can also mutate, including the O and P groups, and eventually lead to a higher number of infections,” said Daniel Sauter, the first author of the PLOS Pathogens study. Sauter points out that these strains are no more aggressive than the pandemic HIV-1 group M strains. The acquisition of an effective tetherin antagonism may however lead to the emergence of viral variants, potentially with increased transmission fitness. However, it still remains unclear why the Vpu proteins of the N strains have only developed modest anti-tetherin activity and why they have lost the ability to degrade CD4. Moreover, it is not yet known whether group N Vpu proteins are also able to antagonise other viral defence proteins (NTB-A and CD1) in the same way as pandemic HIV-1 group N strains. 

Knowledge with some reservations

Prof. Frank Kirchhoff is an internationally renowned AIDS researcher. © University of Ulm

As plausible as the researchers’ findings are, Kirchhoff nevertheless is well aware that the findings cannot be substantiated without suitable models. HIV-1 groups differ from each other and if these differences are able to plausibly explain why one strain has led to a large number of infections while others have not, then the researchers can speculate further. Although Kirchhoff’s discovery that only the Vpu protein of group M is fully functional is regarded as a plausible explanation for the pandemic spread of the HIV-1 M strain, he is nevertheless careful not to draw premature conclusions. “At present we can only speculate that the Vpu protein played a pivotal role in the pandemic spread of the group M viruses. We do not know for sure,” said Kirchhoff.

Kirchhoff is also interested in whether a factor like the Vpu protein that affects the release of viruses might also have an influence on the viral load of genital fluids. The disease is sexually transmitted in over 90 percent of cases. Kirchhoff explains that the effective distribution of the virus depends on the number of viruses in sperm or vaginal fluids. However, it is very difficult to prove this assumption. Sperm samples of men infected with M, N, O and P viruses would need to be taken and the number of viruses determined. However, such analyses require a large number of patients, and, logically, these are not available for rare viruses. 

The greatest obstacle: tetherin

The adaptation of the viruses to their hosts is essentially the result of chance. Although viruses are generally very variable, not all have the ability to switch off the restriction factor tetherin and similar constituents of the human immune system. The fact that only the HIV-1 M group managed to do so shows that highly variable retroviruses are nevertheless not able to do everything. 

Humans have developed a number of defence strategies against such viruses that have been quite successful: eight percent of our genome consists of endogenous retroviruses that have been integrated into our genome and are passed on from generation to generation. The silencing of retroviruses is one way to evade a viral attack, peaceful coexistence is another fairly effective one. AIDS researchers have observed that some monkeys do not become ill despite being infected with human HIV equivalents. They assume that the simian immunodeficiency viruses do not activate the monkey’s immune system to such a great extent and are therefore not eliminated. “As it is not so long ago that the virus jumped to humans, it has not yet been able to adapt efficiently to its human host,” said Kirchhoff explaining the evolutionary dimension. He also believes that this disadvantage is a relatively minor one in view of the fact that it takes infected humans up to eight to ten years before they develop typical AIDS symptoms. 

From Nef to Vpu

People like Frank Kirchhoff who are investigating the causes that lead to the pandemic spread of AIDS also need to take into account evolutionary aspects. Chimpanzees, from which the pandemic HIV-1 group M virus jumped to humans, are our closest relatives. Before the viruses jumped from chimpanzees to humans and gradually adapted to their human host they had already switched off defence mechanisms that are also found in humans. “The situation is somewhat different with tetherin. Simian immunodeficiency viruses (SIV) use Nef instead of Vpu to switch off tetherin in monkeys.”

Frank Kirchhoff also discovered that the HIV-1 Nef protein has become less efficient as a tetherin antagonist in humans. Pandemic group M viruses therefore use Vpu instead of Nef to switch off tetherin. The Vpu protein has somehow ‘learned’ to bind to human tetherin; it either degrades it or keeps it away from the areas where the virus is released.

Four to six simultaneous alterations

Evolution of HIV-1. It is generally agreed that SIVcpz is the cause of the HIV-1 pandemic. This chimpanzee virus is a recombinant of precursors of viruses that are still found in red-capped mangabey and Cercopithecus monkeys. It is assumed that the viruses recombined around 10,000 years ago and subsequently spread to gorillas and humans. The Nef-mediated tetherin antagonism is shown in green, the Vpu-mediated one in red. The dashed line indicates that it is not yet known whether HIV-1 O group strains originated from chimpanzees or gorillas. © Kirchhoff

Retroviruses like HI viruses use the enzyme reverse transcriptase to replicate their genome. This enzyme makes around 10,000 times more errors than human polymerases. Every virus that is produced has on average one modification more than the virus from which it originates. “In an untreated HIV-1 patient with a virus load of between 10 to 100 billion viruses and a viral generation time of one to two days, evolution happens in fast motion,” said Kirchhoff who assumes that four to six amino acid exchanges turned the Vpu protein into a tetherin antagonist.

Only very few differences between pandemic and non-pandemic HI viruses are known. They infect the same cell types and also have many similar characteristics. It is therefore difficult to infer a causal relationship between virus group and pandemic spread. Researchers are working hard to substantiate their speculations with cell culture experiments. Experiments involving monkeys are very rare, very expensive and also ethically highly questionable.

Viruses have a huge genetic variation; even HIV-1 viruses differ in about 30 percent of their amino acid sequences, and this percentage is increased when they are compared with their precursors. Variable genome regions differ from each other to a greater extent than conserved areas and might differ up to 60 percent. Rather than affecting viral function, many nucleotide exchanges and amino acid changes can be seen as a manoeuvre by the virus to evade the human immune system. This also makes it very difficult to identify the one or two mutations from a total of 40 to 50 Vpu protein differences that are able to turn off tetherin. 

Examining natural antiviral factors

The high mutation rate, which is the result of the extremely faulty retroviral replication process, increases the likelihood that HI viruses may develop resistance to drugs. It is therefore worth taking a detailed look at the human immune system’s natural antiviral factors, including tetherin. Kirchhoff’s team has received funds from the European Research Council for a combined evolutionary and proteomics approach to the discovery, induction and application of antiviral immunity factors. This approach complements Kirchhoff’s efforts to find out what enables viruses to spread effectively in the human population. All this knowledge could potentially be used to find ways to counteract the spread of the HI virus.

Data published by the World Health Organisation in November 2012 clearly show that the work of researchers like Frank Kirchhoff and his colleagues is urgently needed: over the last 30 years, AIDS has caused an estimated 25 million deaths; in 2011, 34 million people were infected with HIV and only 25% are receiving treatment; an estimated 1.7 million people died of AIDS in 2011.

Kirchhoff, F.: Humanes Immundefizienz-Virus (HIV), in: Doerr et al., Medizinische Virologie. Stuttgart 2010, p. 315ff.

Sauter, D. et al.: Human Tetherin Exerts Strong Selection Pressure on the HIV-1 Group N Vpu Protein. PLoS pathogens.https://www.gesundheitsindustrie-bw.dewww.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003093

Sauter, D., Specht, A., Kirchhoff, F. : Tetherin: Holding on and Letting Go, Cell 141, April 2010, doi: 10.1016/j.cell.2010.04.02

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