Feature: Moving target

By Fiona Wylie
Monday, 09 July, 2012

In the early 1990s “HIV” became a much maligned addition to the colloquial lexicon due to the sudden emergence in the 1980s of Acquired Immune Deficiency Syndrome (AIDS), the deadly infectious disease caused by the virus.

While not generally front-page news these days, HIV remains one of the most devastating viral pathogens in human history. It is the cause of a continuing global pandemic which, as with many infectious diseases, is having its most severe impact in some of the poorest countries that are the least equipped to manage it.

To date, more than 30 million people around the world have died of AIDS-related diseases, 34 million people are living with HIV, and about 2.7 million new HIV infections are recorded by the World Health Organization each year.

The main factor in HIV’s sudden but persistent rise to infamy – and the reason that we still do not have a vaccine or curative treatment against AIDS – is the virus’s signature ability to diversify rapidly, to hijack host cell machineries and to hide itself from host cell detection.

Professor Johnson Mak from Deakin University in Geelong has made a career investigating the molecular machinery of HIV since he completed his PhD in 1996. His research mixes molecular virology, which was his first research passion career-wise, together with the latest in cell biological imaging and more than a pinch of protein biochemistry to try and decipher what makes HIV so evasive and so devastatingly effective.

Mak will be giving the Frank Fenner lecture at the 2012 Australian Society of Microbiology meeting to discuss his work with this chameleon virus.

As the name implies, molecular virology focuses on the genetics of a virus and viral population over time, and during infection. Mak’s work in this area involves various approaches and techniques, including genomic sequencing, mutagenesis and a novel ‘marker’ system to track how this virus evolves.

“In one example of recent work, we are looking at two mechanisms of evolution for HIV-1: mutation, the transcriptional errors during viral replication; and recombination, the shuffling of genetic material between and within genomes.

“In HIV, recombination occurs much more frequently than mutation, and is a major determinant of the viral diversification that eventually contributes to immune failure and progression to AIDS.”

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It also most certainly plays a major role in viral escape from both the antibody and cellular immune responses of the infected individuals, as well as the generation of multiple drug resistance by shuffling pre-existing mutations in the viral population. “Measuring mutation and recombination rates within the HIV genome is therefore fundamental to our understanding of HIV,” he says.

“The reason that we are so interested in these mechanisms and why this is so important for HIV infections is basically the rate at which it happens. For instance, if I get infected today by HIV-1, within six years the number of different HIV viruses in my body will be far greater than the global diversity of the common Influenza A virus in any given year.

“You can therefore imaging the kind of pressure that puts on our immune system to try and deal with so much viral diversity and different immune targets developing all the time during the same infection.”

Novel markers

As with any good detective story, the methodology must be robust and systematic, and Mak sees sequencing as the most accurate way to interrogate these events of HIV mutation and recombination across virus populations.

In terms of analysing enough samples to get at the required rates of genetic alteration known to occur in HIV, his group has gone to mass-data techniques, including next-generation sequencing.

“We therefore work closely with immunologists and bioinformaticians on these aspects of our research. In particular, Professor Miles Davenport from UNSW has been a very important and long-time collaborator. Over the last few years, we have developed a novel experimental system that allowed us to measure the mutation and recombination rates of HIV directly from the genome for the first time.”

Recombination rates were previously analysed using reporter systems that inserted genes encoding non-viral fluorescent proteins into the viral genome. While offering good in vitro estimates and technological advantages, working with modified HIV genomes may not accurately reflect the level of recombination occurring within a patient infected with HIV. Additionally, recombination would not be detected in regions where the parental genomes are identical, and this effect is often ignored.

“Our novel gene marker system entails introducing modifying each viral genome to create ‘marker’ points that can be followed reproducibly and independently of some of these factors that limit the value and impact of the previously favoured reporter systems.”

Using Mak and Davenport’s system, HIV recombination events can now be measured and compared accurately, even those occurring between closely related genomes as found in the ‘quasi-subspecies’ of an infected individual.

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The analysis is carried out on primary blood cells generated from Red Cross leftovers. Patient samples are next on the list, but initially the team needs to do their work and set the baselines on uninfected cells, with infections carried out in the lab culture dishes under tightly controlled conditions of viral strains and dose etc.

“Having said that, we are also currently doing some in vivo work in collaboration with Steve Kent at Melbourne University on monkey models of SIV [simian immunodeficiency virus] infection, although talking about that will be for another time.”

Cellular personalities

Mak’s group is also looking at those recombination and mutation events that are dependent on the viral target. “HIV infects two immune cells – T-lymphocytes and macrophages – and we have started to find marked differences in recombination and/or mutation rates between these cell types that might at least partly explain how HIV evolves.”

We already know that HIV-infected T-lymphocytes produce a lot of virus, but they also die off very quickly. On the other hand, macrophages carry much lower numbers of viral particles, but live for longer once infected. Indeed, macrophages are often referred to as a ‘reservoir’ of the virus.

“However, because the rate of HIV diversification is way higher in macrophages than in T-lymphocytes, we believe that somehow the cell longevity allows to virus to change genetically and continue to pump out different types of virus before it dies, enabling a higher rate of evolution as the macrophage infection proceeds.”

Another thrust of the Molecular Virology group that Mak will touch on in the Fenner lecture surrounds some small-molecule inhibitors under development. These are designed to slow down that evolutionary process and thus also limit the rate by which HIV changes in the body.

“Our hope is that such molecules could eventually be used clinically to reduce the pressure on the immune system and allow it the time to hold back the viral tide and deal with the infection,” he says.

Such a strategy should also impact on anti-HIV vaccines, because as it stands now, vaccines – and, indeed, any antiretroviral therapies – have to cope with a virus that is so rapidly changing that any agent is basically not fast enough to help the immune system fight and resist the virus.

“So, our inhibitors could potentially help both the clinical treatment and vaccine approach for fighting HIV.”

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