Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are the major causes of viral lower respiratory tract infections (LRTIs). There are currently no licensed vaccines or antivirals for RSV and hMPV; and so research is directed towards the development of live-attenuated vaccines. One pathway to attenuation is to disrupt the nucleocytoplasmic transport of structural proteins of a virus. The nuclear localisation of a structural protein is a strategy used by many viruses to inhibit host antiviral responses. RSV matrix (M) protein shuttles between the cytoplasm and the nucleus, with nuclear localisation occurring early in infection (around 18 – 24 hours post infection) and cytoplasmic localisation later. The disruption of RSV M nuclear import decreases the titre of infectious particles while the disruption of nuclear export ceases the production of infectious particles. The nucleocytoplasmic transport of RSV M is regulated by CK2 phosphorylation although the detailed mechanism is not well understood. RSV M(205) (T),which is adjacent to the nuclear export signal, has been predicted as a putative regulatory site. The nucleocytoplasmic transport of hMPV nucleocapsid (N) protein, however, has never been described. The hMPV N has been shown to localise in the nucleus late in the replication cycle (day 5 post infection),but further research is required to fully understand the mechanism. Published literature (RSV M) and unpublished results (hMPV N) from the Ghildyal group led to a hypothesis that disruption of the nucleocytoplasmic transport of a viral structural protein can lead to attenuation. In this study, two hypotheses were tested: (1) mutation in RSV M205 reduces the production of infectious progeny and the induction of pro-inflammatory responses; and (2) the hMPV N has inherent nucleocytoplasmic transport ability. The hypotheses were tested in two aims. Firstly, to investigate the role of the RSV M(205) (T) in virus growth and induction of pro-inflammatory response in Vero E6 and A549 cells; and secondly, to investigate the nucleocytoplasmic transport ability of hMPV N in transfected Cos-7 cells. To understand the role of RSV M(205) (T),cells were infected with recombinant RSV strain A2 having threonine (T) substitution to alanine (A) at M205 (rRSV A2 M(T205A)). Viral titres and expression of IL-8 and RANTES were determined using plaque assay and ELISA, following single cycle and multiple cycle replication assays. To define hMPV N nucleocytoplasmic transport, full-length and truncated hMPV N constructs fused to green fluorescent protein were expressed in transfected Cos-7 cells. rRSV A2 M(T205A) was found to be capable of infecting Vero E6 and A549 cells, but was unable to transmit the infection between cells. The inability to spread leads to a reduced induction of IL-8 and RANTES expression, which was significantly suppressed in the interferon (IFN)-α/β-producing A549 cells, compared to the wild type rRSV A2 M205 (T). hMPV N was found to have inherent ability of nucleocytoplasmic transport, with a potential nuclear export signal (NES) identified at amino acid 1 – 15 and a region containing a nuclear localisation signal (NLS) identified at amino acid 192 – 250. This study shows for the first time that the RSV M(205) (T) is an important site for the success of transmission of infection and RSV M has a role in the suppression of IFN-α/β. This study was also the first to show that hMPV N is capable of nucleocytoplasmic transport, with potential nuclear transport motifs have been identified. By describing the nucleocytoplasmic transport function of the RSV M and hMPV N proteins, this study contributes to attempts to develop live-attenuated vaccines for RSV and hMPV.
|Date of Award
|Reena Ghildyal (Supervisor) & Michelle Gahan (Supervisor)