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Science Technology & Engineering |
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Dr Jason Mackenzie and the
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Our overall objectives are to investigate and unravel the replication mechanism of two positive-stranded RNA viruses (West Nile virus [a flavivirus] and Mouse Norovirus [a Norovirus]) that are highly pathogenic to humans and cause outbreaks of encephalitis and gastroenteritis. Our aims are to determine how and where these viruses replicate within infected cells and what host components/organelles are “used and abused” by the virus. We aim to correlate this abuse of host with the pathogenic outcomes associated with viral infection. In conjunction with these studies we are investigating how viruses can evade our immune system and in particular how viruses can bypass the antiviral activities of our first line of defence; the innate immune system.
In particular we investigate:
Our current research has sought to evaluate the role of cellular lipids in flavivirus RNA
replication and membrane induction. We have observed that a host protein regulating
cholesterol biosynthesis is upregulated during infection and redistributes to the sites of
virus replication. Additionally compounds affecting the cells capacity to produce and recycle
cholesterol have differing effects on virus replication, with drugs affecting cholesterol
biosynthesis having the most profound effects. One consequence of the redistribution of
cholesterol is the apparent dissociation of lipid raft molecules. This consequence has
implications relating to cellular metabolic pathways including immune activation cascades.
In collaboration with Rob Parton (Institute of Molecular Biosciences).
We have previously identified the protein composition and roles of unique cytoplasmic membrane
structures that are induced upon flavivirus infection. These membrane structures appear
crucial to the efficient replication of flaviviruses and are intimately linked to the
exponential increase in virus production. These membranes can be easily identified with
antibodies with both the light and electron microscopes, however these are static
representations. Recently we have identified the viral protein responsible for these membrane
changes and thus we can directly target this protein for analysis. We aim to utilize the green
fluorescent protein and time-lapse epi-fluoresence to visualize the formation and
proliferation of virus membranes over real-time in living cells. To this end we have
constructed in-frame insertions of the GFP gene into the Kunjin virus infection clone and
replicon. Analysis of these constructs after expression reveals that they are defective for
replication. Fortunately though they can be rescued with a helper replicon to produce and
express the GFP-fusion proteins. Currently we are investigating techniques to provide us with
viable constructs in the future.
In collaboration with Gareth Griffiths (EMBL, Heidelberg, Germany).
In response to infection by pathogens our cells and body produces proteins that fight and combat the invading pathogen. The production of such “anti-viral” proteins is tightly regulated though, primarily by chemicals known as interferons. One of these antiviral proteins is MxA. MxA has broad spectrum antiviral properties against many viruses, in particular viruses similar to influenza and measles viruses. One of our aims was to assess whether MxA could also impart these antiviral activities against flaviviruses. Therefore we observed whether over-expression of MxA, independently of intereferon, could protect cultured cells against flavivirus infection. Analyses revealed that either flavivirus RNA replication or virus production was hampered by MxA expression. This evasion does not appear to be due to a viral-encoded antagonist, although an unknown host protein does appear to specifically associate with MxA during infection. The role and identity of this protein is currently under investigation. Interestingly some of our data has indicated that the prolific membrane rearrangements and rapid flavivirus assembly process may “hide” the viral components from MxA and other host surveillance proteins thus preventing the host cells from stimulating protective mechanisms.
Like other +ssRNA viruses, MNV-1 replication is closely associated with host membranes, which undergo significant rearrangement during infection. We have characterised the localisation of the MNV-1 non-structural proteins, and show that the MNV-1 replication complex initially associates with the centriole and later with membranes derived from the ER. The association of MNV-1 replication with the centriole early on in infection, indicates a possible role for microtubules in the migration of virus to the site of replication. By assessing the effect of cytotoxic drugs on infected cells, we have additionally demonstrated a role for actin in the release of newly formed virus from cells.
Content approved by: Jason Mackenzie
Page maintained by: Craig Lighton
Last updated: 11 June, 2008
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