Overview:

This unit was established in February of 2006 when Dr. Kirsten Spann joined SASVRC/CMVC from the National Institute of Allergy and Infectious Diseases, NIH, USA. The long-term vision of the unit is to develop antiviral treatments for paediatric respiratory viruses, and to contribute to the world-wide effort to develop effective vaccines for hRSV and hMPV. The unit is focused on Respiratory Syncytial Virus (RSV) and human Metapneumovirus (hMPV).

HRSV is the principal cause of severe lower respiratory tract disease in infants and young children, and can also cause serious disease in immunocompromised adults and the elderly. Clinical manifestations vary from rhinitis and otitis media, to bronchiolitis and pneumonia. The global annual RSV infection rate is estimated to be 64 million, with approximately 160,000 cases resulting in death. The hospitalization burden due to RSV infection in Australia is considerable. Various research efforts are underway to develop vaccines to combat RSV. However none have been licensed. Monoclonal antibody prophylaxis and treatments have been moderately successful, but are expensive. There is a need to explore alternate antiviral treatments for RSV. To this end, we are interested in investigating the mechanisms of host innate immune evasion of RSV.

RSV is also a principal cause of asthma exacerbation and plays a role in the inception of asthma along with other environmental and genetic factors. It is believed that asthmatics have defects in their innate antiviral response to viral infections, which makes them more susceptible to the spread of viral infections from the upper to the lower respiratory tract. hMPV is also a trigger of asthma exacerbation, although to a much lesser extent. We are interested in investigating the mechanistic differences in the innate immune response of respiratory epithelial cells from asthmatic and non-asthmatic subjects.

HMPV is a significant cause of both upper and lower respiratory disease in infants and children. The clinical symptoms are similar to those of RSV and by age 5 virtually all children have been exposed to hMPV. In Australia, hMPV-related disease occurs in late winter to early spring with an incidence between 15-30%. There are currently no vaccines or specific treatments for hMPV infection. We are interested in identifying potential mechanisms by which hMPV evades the innate immune response and the development of siRNA molecules as antivirals.

Research Projects:

Mutation of the elongin C binding region of NS1 results in the degradation of NS1 and attenuation of RSV.
Kirsten Spann, Claire Straub, Wei Har Lau, Maxine Preston in collaboration with Jeff Gorman (QIMR) and Peter Collins (NIAID).
Funding support: The University of Queensland, Royal Children's Hospital Foundation, NHMRC

The only functional region of NS1 currently hypothesized, is a putative elongin C binding domain. It has been proposed by Elliot et al., (J. Virol. 2007, 81(7): 3428-3436), that NS1 forms an E3 ligase complex similar to SOCs and VHL proteins to target STAT2 for ubiquitination and degradation via the proteosome. NS1 does contain a domain that shares homology with the elongin C binding domains of SOCs and VHL proteins via which they form an E3 ligase. We have mutated this region within NS1 in live infectious virus by replacing the 3 homologous residues with alanine and performing reverse genetics.

This mutation attenuated RSV in both Vero (type I IFN incompetent cells) and A549 (type I IFN competent cells), suggesting that attenuation was partially, but not exclusively the result of the type I IFN response to infection (Fig. 1). Attenuation in A549 cells was also similar to that observed when the entire NS1 protein was ablated (rRSVΔNS1). Quantitative PCR to detect RSV mRNA and IFN-β mRNA showed that this mutation resulted in a significiant induction of IFN-β, either greater than or similar to deletion of the entire NS1 protein. Immunofluorescent and Western blot detection of NS1 using either anti-NS1/2 polyserum or a monoclonal to the FLAG tag demonstrated that mutation of the putative elongin C binding domain resulted in the degradation of NS1 protein over time (Fig. 2). This suggests that this region is important to the survival of the NS1 protein and would explain why attenutation of the mutant is similar to deletion of the entire NS1 protein.

Immunoflourescence demonstrated that the NS1 protein was sequestered within vesicles, or accumulated in the cytoplasm when the elongin C binding domain was mutated. Co-localisation with LAMP1 and not other organelle markers suggests that these vesicles are associated with lysosomes and that NS1 is degraded via a lysosomal-directed mechanism (Fig. 3a). Western blot analysis using a marker for autophagy, the anti-LC3B protein antibody, demonstrated that wild-type RSV induced autophagy, although mutating the elongin C region did not (Fig. 3b).

Figure 1. Mutation of the elongin C binding regions of NS1 (rRSVNS1F-ELCmut) attenuated the virus in both (a) A549 cells and (b) Vero cells compared to unmodified RSV (rRSVNS1F). Figure 2. Both (a) Western blot analysis and (b) immunofluorescence demonstrate the degradation of NS1 when the elongin C binding domain is mutated (rRSVNS1FELCmut). Figure 3. (a) Immunofluorescent detection of NS1 in Vero cells infected rRSVNS1F-ELCmut (mutated NS1), showing the co-localisation of NS1 with LAMP1 suggests lysosomal association. (b) Autophagy was not induced in rRSVNS1F-ELCmut-infected cells.

The original hypothesis to be tested was that mutating the elongin C binding domain of NS1 would disable the degradation of STAT2 in RSV-infected cells. Due to the degradation of NS1 by this modification, we were not able to test this hypothesis, although we did demonstrate that in wild-type RSV infected cells, STAT2 was completely degraded, whilst in ΔNS1-infected cells, not only was STAT2 not degraded, it was also activated and translocated to the nucleus. In rRSVNS1F/ELCmut-infected cells, there was an intermediate effect on STAT2 in that STAT2 was not degraded, but was not obviously activated either (Fig. 4). This is expected given an intermediate level of NS1 expression.

Figure 4. STAT2 degradation was observed in wild-type RSV infected cells, but not ΔNS1 or rRSVNS1F/ELCmut-infected cells.

The functional significance of the C-terminus of the NS1 protein of RSV.
Kirsten Spann, Claire Straub, Qiao Ye Tan, Angela Dougall, Maxine Preston in collaboration with Jeff Gorman (QIMR) and Peter Collins (NIAID).
Funding support: The University of Queensland, Royal Children's Hospital Foundation, NHMRC

Recombinant RSV vaccine candidates which contain deletion of the entire NS1 gene are being considered for development for clinical trials. However infection studies in primates suggest that full NS1 deletion, as is the case for full NS2 deletion, may be overattenuating . NS1 has functions other than interferon modulation. For example, NS1 suppresses early apoptosis in infected cells and deletion of NS1 results in an apoptotic phenotype that makes the propagation of virus difficult. Once the specific regions of NS1 involved in IFN suppression, and other functions, are identified, these may be deleted or modified in live recombinant viruses, leaving the remainder of NS1 intact. This may improve the immunogenicity of a vaccine candidate. Apart from the putative elongin C binding domain, no other functional regions of NS1 or NS2 are known. Despite NS1 and NS2 both functioning as type I IFN antagonists, the only region of homology between them is the 4 residue C-terminus: DNLP. We have replaced this region with alanines (AAAA), in both NS1 and NS2 using site-directed mutagenesis and reverse genetics. We have also generated block deletions of the C-terminus of NS1 to generate a suite of mutants. These mutant rRSVs also express the FLAG-tag on NS1 and the c-myc tag on NS2 for purification and identification:

rA2 – no tags
WT(F/C) – NS1-FLAG and NS2-c-MYC
NS1Cterm alanine replacement
NS1Δ10 residues from the C-terminus
NS1Δ20 residues from the C-terminus
ΔNS1

All viruses have been validated by sequencing and also by western blot analysis of protein expression. The plaque phenotype of the viruses and the effect of NS1 C-terminal modification on viral growth has been investigated. Deletion of either 10 or 20 residues from the C-terminus of NS1, or alanine replacement of the 4 x C-terminal residues, attenuated RSV growth to levels intermediate between WT and ΔNS1 in A549 cells capable of an interferon response. These C-terminally-modified rRSVs were less attenuated in Vero cells, which do not secrete type I or III interferons, suggesting that the C-terminus is involved in suppression of the interferon response. The correlation between attenuation of viral growth and the interferon response is currently being investigated. The plaque phenotype was also affected by C-terminal modification in that deletion of 10 or 20 residues from the C-terminus of NS1 resulted in a smaller plaque phenotype than WT RSV, although plaques were not as small as those formed by the NS1 virus (Fig. 5). This is also indicative of slower viral replication and spread.

We have also found that modification of the C-terminus of NS1 effects the expression of NS1, in that there is a marginally significant reduction in the amount of NS1 expressed when the C-terminus is modified. This will need to be taken into account when the effects of C-terminal modification on cell responses are interpreted. Any mutants that look promising for inclusion in current vaccine candidates will be further characterized for attenuation in vivo. Ultimate inclusion of mutations identified here as significant for a type I IFN response to RSV infection will be included in current vaccine candidates in collaboration with Peter Collins (NIAID).

 

Figure. 5. Differences in the plaque phenotype of wild-type RSV (rA2 and WT(F/C)) and the different NS1- modified rRSVs.

RNA interference of the G gene of hMPV
Kirsten Spann, Maxine Preston, Claire Straub in collaboration with Suresh Mahalingam (University of Canberra) and Ralph Tripp (University of Georgia).
Funding support: NHMRC.

RNA interference (RNAi) is a gene-silencing mechanism in which small dsRNA molecules target cognate RNA for destruction, causing post-transcriptional silencing. Synthetic siRNA drugs for several viruses (HIV, RSV, SARS) are in clinical trials. We are investigating the potential of the hMPV G attachment protein as a targets for RNAi therapy, as the G protein is required for attachment and has also been implicated as a type I interferon antagonist
We have established a non-toxic siRNA transfection method for in vitro studies, and identified siRNA molecules that successfully knock-down expression of the G gene by 80-90%, as demonstrated by qPCR to quantify mRNA expression (Fig. 6a).

Figure 6. (a) Knock down of G by the siG2 molecule. (b) G knock-down did not attenuate growth of hMPV in A549 cells.

Knock-down of G did not attenuate hMPV in A549 cells (Fig 6b), nor did it result in a significant increase in the type I IFN response to infection in A549 cells. This suggests that G is not required for in vitro replication nor is it a type I IFN antagonist. This is interesting as G has been proposed as an antagonist in the literature. hMPV also failed to suppress induction of type I IFN by poly I:C, a known exogenous inducer, suggesting that if hMPV does encode an IFN antagonist, it is not highly effective.

Despite the evidence that G is not a type I IFN antagonists, it is still required for replication in vivo and therefore a viable target for antivirial RNAi. The effects of G gene knockdown on viral replication in a mouse model is currently being investigated.

The differences in the innate antiviral response to RSV infection of epithelial cells from asthmatic and non-asthmatic children.
Kirsten Spann, Maxine Preston, Claire Straub in collaboration with Peter Sly and Emmanuelle Fantino (QCMRI).

Respiratory viruses first invade the upper airway and then may progress to infect the lower airway. Increasing data suggests that asthmatics may be less able to prevent this spread, related to a decrease in the anti-viral innate immune response in their respiratory epithelial cells. We do not know if this defect is equally present in the upper and lower airway cells or whether this contributes to acute viral-associated exacerbations of asthma. In this project we will identify differences in the innate immune response of airway epithelial cells from asthmatic and non-asthmatic children to viral infection.

We have collected nasal (upper) and tracheal (lower) epithelial cells from children from 2-10 years admitted to the RCH for elective surgery. Once a critical number of subjects have been selected, they will be grouped into atopic asthmatic, atopic non-asthmatic, non-atopic asthmatic and non-atopic non-asthmatics, however for the moment the study is blinded. The cells are grown in culture and infected with RSV A2 lab strain. The innate immune responses to these viruses and viral replication will be quantified. At this stage we have collected data on the induction of type I (a/b) and type III (l) interferon. In the near future we aim to also collect data on the secretion of pro-inflammatory cytokines and other relevant measured of antiviral response. Viral replication has also been quantified by titrating virus shed from infected cells. The kinetics of high and low dose is being investigated by infecting cells at either a high (MOI 3) or low (MOI 0.1) multiplicity of infection. In the near future we will also infect these cultured with hMPV.

The primary cell culture system developed here will allow the characterisation of differences in innate immune responses to viral infection of airway cells from asthmatic and non-asthmatic children in a controlled in vitro system. We will also establish if less invasive nasal brushing can be used as a surrogate for lower respiratory cells, and allow immunological studies during exacerbation. Mechanistic studies in children, such as this, will be important to better understand how we can develop new preventions and treatments aimed at reducing the significant cost and morbidity associated with asthma.