Viral secrets revealed bit by bit

Professor Jeffrey Gorman established the Protein Discovery Centre at QIMR (QPDC) in early 2006 with the overall aim of chemically identifying and analysing proteins involved in physiological and disease processes. It also aims to decipher the ways in which these proteins interact functionally in vivo.

The centre is equipped with an array of the latest high-performance mass spectrometers and other state-of-the-art ancillary equipment, allowing the most up-to-date protein chemistry and proteomics approaches to be applied.

This involves a range of techniques from amino acid sequencing of isolated proteins digested from polyacrylamide gels through to identifying the ways in which the proteins are modified post-translationally to function. This combination of expertise and infrastructure makes the QPDC increasingly able to address important and otherwise intractable biological questions.

One of the QPDC’s main project areas is deciphering viral/host protein interactions, involving a three-year collaboration with Kirsten Spann at the Sir Albert Sakzewski Virus Research Laboratory (SASVRL) at the Royal Children’s Hospital in Brisbane and Peter Collins at the National Institutes of Health in the US.

Collins is an experienced virologist and a world authority on paediatric respiratory viruses, and in particular respiratory syncytial virus (RSV). He was the first person to make a recombinant clone of RSV and is currently pursuing gene knockout experiments with the aim of producing a vaccine.

A graduate of the University of Queensland, Spann worked in Collins' lab in the US as a postdoctoral fellow before returning to Australia and setting up her own group in Brisbane, literally just down the road from Gorman.

“It is a very good collaborative network,” Gorman says. “Cell lysates from the gene knockouts experiments come from the US for analysis in Brisbane at SASVRL and QIMR, and it is working really well.”

RSV is a major cause of severe lower respiratory tract infection in infants and serious disease in immunocompromised adults and the elderly. The health care burden due to RSV infection in Australia is considerable – between half and two per cent of children with their first RSV infection require hospitalisation, with most of these kids under six months of age.

Nearly all children will have been infected with the virus by two to three years of age. However, natural infection with RSV does not induce protective immunity, and thus individuals can be infected multiple times even within the same season. Development of an effective and safe RSV vaccine is a high research priority.

---PB--- Host-cell proteins

The long-term vision of Gorman’s collaboration with Spann and Collins is to develop antiviral approaches for paediatric respiratory viruses for which vaccines are not yet available, starting with RSV.

The team is using sophisticated genetic approaches combined with basic virology and Gorman’s protein-analysis expertise to decipher exactly how RSV negates the host innate immune response.

Specifically, the team wants to define crucial interactions between RSV and host-cell proteins during infection and assembly in conjunction with mass spectrometric analysis. Emerging findings have the potential to inform both therapeutic drug design and preventative vaccine approaches.

Scientists in this field are increasingly aware that viral proteins can interact in a variety of ways with cellular proteins during viral replication in the host cell, rather than only during the initial stages of infection. These interactions are often required for efficient viral replication and thus important for pathogenesis.

Recent technological advances in protein analysis have increased the prospects for addressing this area, with protein experts like Gorman now better able to identify molecules and to detect protein interactions in low quantities and in complex mixtures such as virally infected cell lysates.

All viruses produce specific proteins that inhibit the cellular immune response, thereby setting up a clearer path for increased infection. RSV is known to interfere with the induction and amplification of interferon activity in particular, through the action of two non-structural proteins of the virus, known as NS1 and NS2.

“Kirsten has been interested in how NS1 and NS2 act for a long time, and together we are trying to identify direct interactions between these two viral proteins and proteins of the host cell, as well as the structural drivers for the interactions in terms of protein domains,” Gorman says.

“At the same time, Peter’s lab is oriented towards finding vaccines to combat RSV. Their recent focus is knocking genes out to come up with the most attenuated virus that will still function as a vaccine.”

For instance, if the structure of NS1 or NS2 is overly disrupted, RSV cannot function well enough to serve as a live-virus vaccine. Of course, there are successful live-virus vaccines for other members of the same virus family (Paramyxoviridae) such as mumps and measles.

“However, when the original approaches used to generate those vaccines were used for RSV, it actually created more problems than if the children became naturally sick,” he says.

“So, vaccinating naïve kids made them more susceptible to subsequent natural infections, and there were some deaths implicated with that vaccination program. The problem was tracked down to a piece of one of the viral proteins that seems to exacerbate the immune response to the virus, causing an infiltration of eosinophils into the lungs. Of course that put the efforts back many years and makes clinical trials more difficult to establish.”

---PB--- Knockout analysis

The protein analysis of the genetically modified (knockout) viruses generated at NIH is yielding some promising data.

“Using a differential 2D-gel-based proteomic approach, we have determined that one of the RSV glycoproteins strongly represses the expression of a specific host cell protein,” Gorman says.

“This protein also contains a small sequence module that was associated with the adverse vaccine reaction.

“The same effect is not seen in cells infected with wild-type or unmodified virus – it is specific to the knockout. So there is something in the whole virus that prevents the upregulation of this host protein.”

When these specific viral components are not there (knockout virus), particular proteins that form part of the cellular response are turned on.

“We think this is important for combating the virus because this host cell protein is uniquely affected compared to all of the other activated proteins on the gel. By doing a series of limited knockouts, that is, deleting bits of the RSV protein in steps, we just might be able to find out which piece of the knocked-out viral protein is the critical one, and then use that in a subunit vaccine strategy down the track.”

Knockouts of the NS1 and NS2 proteins of RSV generated in Spann’s laboratory are also producing interesting results with respect to repression of the same key host protein regulated by the viral glycoprotein. Gorman and Spann are currently pursuing this by tagging the NS proteins for affinity-based pull-down experiments to identify specific binding partners for NS1 and/or NS2.

“It will be interesting to see if those experiments will tell us more about the different cellular pathways involved with immune suppression,” Gorman says. “We can also do things such as use cultured cells that lack the interferon pathway, and see what proteins are then pulled down by the different RSV proteins of interest such as NS1 and NS2.”

---PB--- Flat out LC-MS

The workhorses of Gorman’s RSV analysis are two Orbitrap liquid chromatography-mass spectrometers, one of which uses electrospray ionisation while the other is fitted with MALDI ionisation. The ones at QIMR were the first installed in Australia and have been going flat-out ever since.

Gorman describes these high-performance instruments that take up little more bench space than a large centrifuge as a piece of sheer ingenuity. “These are the top-line commercial instrument for digging deep and digging very specifically. They provide very high mass accuracy, very good sensitivity and very fast data acquisition throughput.”

Typically, the team also acquires mass spectrometry data with an older MALDI TOF-TOF instrument, so two different sets of data are pointing at the same problem to ensure that crucial structural features of the proteins do not escape detection due to a lack of sensitivity with a specific technology.

Gorman has just placed an order for one of the next generation of mass spectrometers coming from Bruker and is eagerly awaiting its arrival.

“We know from preliminary testing of standard mixtures that this new instrument will be able to dig even deeper and more specifically than the Orbitrap. It does this by being simply faster and more accurate. In practical terms this means just seeing a whole lot more proteins at high accuracy and resolution, which contributes significantly to the quality of the data that you can use.”

The improved speed and accuracy also means being able to analyse smaller sample sizes, which is a big bonus for cell biologists who are often dealing with relatively low concentrations of proteins and only small changes associated with function. “Basically, we can now generate significantly larger and more meaningful protein identification and characterisation datasets than we could even just a few years ago.

“We are basically developing specialised methodologies to really push the envelope of analysing interactions between cellular and viral proteins. Those things just wouldn’t happen in an applied research or even basic research environment.

“So, from our perspective, we are really taking this forward, and in doing so, we are also improving our skills for looking at the more medical or biological issues associated with RSV and other viruses.

“From a more local perspective, the Paramyxoviridae family of viruses that contains RSV also includes Hendra and Nipah viruses and approaches developed with RSV may well be applicable for development of control methods for these other nasty viruses.”