HIV proteins and cellular control

HIV is an extraordinarily smart virus. Just as researchers track down another potential vehicle for halting its spread, it throws up another roadblock. Research on vaccines has been ongoing for 20 years, with neutralising antibodies remaining the best hope after the recent failure of Merck’s Phase III trial of an adeno-vectored T-cell vaccine, but at the moment combination antiretroviral therapy is our only weapon.

As HIV has a relatively limited number of its own proteins, researchers have obviously zeroed in on these to try to inhibit viral replication. The virus’s ability to mutate has sidestepped many of these attempts, however, so some are looking at inducing the cellular mechanism known as RNA interference to halt the virus in its tracks by silencing gene expression. Even then, however, HIV might have a solution.

Associate Professor Damian Purcell, who heads the Molecular Virology Laboratory at the University of Melbourne, has been experimenting with RNAi since he first heard that it functioned in mammals at the 2001 RNA Society conference addressed by Tom Tuschl, one of the discoverers of mammalian microRNAs (miRNA) who also found that RNAi functions in mammalian species.

Like many, Purcell rushed back to his own laboratory and began designing short interfering RNAs (siRNA) – the small strands of RNA that bind to complementary sequences of messenger RNAs and silence gene expression post-transcriptionally – as a way of targeting HIV. However, as everyone else discovered, HIV is often able to rapidly escape control by siRNAs, its mutability working in its favour yet again.

That doesn’t mean RNAi may not prove a potent weapon against HIV, as the multitude of clinical studies currently underway illustrate. It’s just that many viruses, especially HIV, have evolved with the ability to counter these cellular control mechanisms, so designing the right siRNA is paramount. short hairpin RNAs (shRNAs) are also in development. For example, in October a trial of an shRNA-based RNAi therapy in HIV patients released promising – although very early – results.

Silencing HIV genes using siRNAs and shRNAs does reduce viral replication and as such is a very promising line of enquiry. Like many others, Purcell’s laboratory has designed lentivectors for delivery of siRNA to target HIV, but his team has also pursued other protein targets, in particular cellular proteins that do not mutate and therefore may provide an alternative route.

One target is the HIV trans-activation response RNA binding protein (TRBP), which inhibits the RNA-dependent protein kinase (PKR) response. TRBP binds to PKR, one of the main interferon-response proteins that shut down the expression of viruses in cells, and in HIV infection keeps the PKR pathway from doing its job.

Purcell’s lab has been studying TRBP for many years and has uncovered some surprising activities of the protein in astrocytes. The researchers have also discovered that TRBP turns out to be an essential component of the miRNA biogenesis pathway – it is a binding partner for Dicer, the protein that cleaves double-stranded RNA into siRNAs and sets off the whole RNAi cascade.

“It has been an interesting observation – almost a roadblock – that the TRBP molecule turned out to be an essential component of the RNAi pathway,” Purcell says. “It turns out that it’s a very good target (for inhibition) but that also eliminates the ability to produce siRNAs.

“There’s a lot that we don’t understand in the control of gene expression at a post-transcriptional level and viruses are very busy in this area. Many of them make proteins that are able to defend against these cellular control mechanisms, and curiously the same viral proteins that inhibit PKR also inhibit RNA interference.”

---PB--- Astrocytes and innate immunity

Our understanding of these viral proteins, and how our own immune system is unable to counteract them, has been much improved by studying astrocytes, the glial cells that for some reason do have a natural resistance to productive HIV infection. In 2005, Purcell and colleagues from the University of Melbourne and from McGill University in Canada looked at how astrocytes manage to silence HIV infection.

No one really knows exactly how it is done, but while astrocytes are infected by HIV, the virus’s RNA is unable to produce proteins in these particular cells. “Astrocytes are naturally infected but curiously the infection goes through all of the steps of entry and integration of the virus, and it even transcribes large amounts of RNA, but those RNAs don’t seem to yield proteins,” Purcell says. “It is blocked at a post-transcriptional level and we thought that was interesting.

“One of the things that seemed to be a candidate at the time was the known interferon response pathways involving PKR. We suspected that TRBP was a binding partner and a cellular control protein for the level of responsiveness for the PKR pathway.”

Astrocytes express unusually low levels of TRBP that makes the unchecked PKR highly responsive to viral RNA helix, prompting a cascade of cellular antiviral responses.

“When we supplied (TRBP) back to those cells at higher levels we could restore efficient HIV production. We don’t know exactly the reason but we suspect that in the brain a lot of these genetic pathways for antiviral responses have to be more sensitive than elsewhere as you can’t afford to have an expanding T cell response in the brain, for example.”

With the discovery that TRBP is a binding molecule for Dicer, and his work on the remarkable ability of astrocytes to fend off HIV by keeping TRBP at low levels, come some fascinating research potentials. “What the research is finding now is a convergence between this innate immune pathway involving PKR and interferon, which we’ve known about for a long time, and how through TRBP this connects to the genetic immune system of RNA interference. What we’ve been trying to figure out is the relative importance of RNAi compared with the PKR-interferon-driven pathway in the control of HIV.”

In addition to looking at some of the cellular proteins, Purcell and his team are of course looking at HIV’s small but powerful complement of viral proteins, as well as the RNA elements that make HIV such a fascinating and challenging virus to study.

Tat Rev, Nef and Vif are intensely studied around the world, as is the envelope, or Env, protein, which is of great interest in vaccine development. “Over the years we have been interested in understanding what virus RNA elements we must retain in order to preserve optimum expression of the viral proteins for our various HIV vaccines, without keeping so much that we allow them to reconstitute an infectious pathogenic agent,” he says.

“We’ve looked at the functions of the small control proteins Rev and Tat, but in recent times our vaccine efforts have focused on envelope, because we are very keen on understanding how we can express many different envelopes and assess them as candidate vaccines for neutralising antibodies, because that seems to be the best hope for an effective HIV vaccine.”

With the limited resources that all Australian researchers must face, Purcell has decided to take a different angle in potential vaccine development – the understanding of how the different RNA elements work to control efficient envelope expression in HIV.

Purcell says that one of the most fascinating aspects of using HIV as a model genetic system is that it must obey the rules of human genetics to survive. “Many of the things that have been observed with post-transcriptional control mechanisms have turned out to also operate in certain cellular genes during later analysis. HIV is a fairly plastic genetic system and is easy to study and manipulate, and has revealed many new mechanisms.”

---PB--- Functional RNAs

Associate Professor Damian Purcell started out in molecular immunology at a time when virology wasn’t exactly booming in Australia. He did his PhD in the early 80s with Ian McKenzie, the former head of the Austin Research Institute, looking at the genetics of cancer and then moving on to endogenous retroviruses.

“Then HIV appeared, right when I was a starting PhD student,” he says. Purcell was working on gibbon ape leukaemia virus and making antibodies against what he thought might be a new human virus, and was collaborating with the now famous Robert Gallo lab at the University of Maryland.

“He was working on these kinds of viruses and I went over to one of the early Keystone conferences in 1985, when they’d just finished their work identifying the HIV virus,” Purcell says. “I wanted to talk to him about what we thought was a human endogenous retrovirus, but he said ‘thank you, but we’re frankly just not interested because HIV is so important. We’ve dropped all that other stuff and we’re just working on HIV.’ He was right.”

Purcell then received a CJ Martin Fellowship and went to the US National Institutes of Health to work with Malcolm Martin, who did a lot of the early molecular biology of HIV. “In that lab they were very interested in RNA elements and the functions of small RNAs, so I’ve been interested in functional RNA since then.”