Abstract
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor.
Highlights
The impact of RAB1A on Vaccinia virus (VACV) replication and spread was examined using VACV-A5L-EGFP, a VACV strain which has the A5 viral capsid protein tagged with EGFP, thereby enabling virus growth to be estimated by measuring fluorescence levels (Beard et al, 2014; Carter et al, 2003)
Onestep growth curves and viral protein expression profiles indicated that, despite its involvement in a wide range of cellular functions that contribute to VACV replication including integrin localisation and actin regulation, RAB1A had a negligible effect on early stages of viral replication with normal amounts of early and late proteins and intracellular mature virions (IMVs) produced in the absence of RAB1A (Fig. 2)
The negative effect of RAB1A depletion on multicycle growth of VACV (Fig. 1A and C) is likely due entirely to the loss of intracellular enveloped virions (IEVs), cell associated virion (CEV) and enveloped virions (EEVs), emphasising the key role previously established for CEVs and EEVs in promoting rapid spread of VACV in cell culture (Blasco and Moss, 1991, 1992), reviewed by Smith et al (2003)
Summary
RAB1A has been shown to be rapidly recruited to plasmamembrane lipid rafts within 30 min of VACV infection (Schroeder et al, 2012), suggesting a possible role in virus entry In support of this theory, RAB1A regulates the recycling of ITGB1 to the cell surface (Wang et al, 2010) and this integrin protein has been shown to facilitate VACV entry (Izmailyan et al, 2012). Knockdown of golgin-97 in VACV infected cells unexpectedly resulted in disruption of IMV formation and accumulation of immature virions (Alzhanova and Hruby, 2006, 2007) It is unclear why this protein, which facilitates a late stage of the vesicle transport system, should inhibit early stages of VACV morphogenesis. Given the multiple plausible hypotheses for the role of RAB1A in VACV replication, we investigated the stage of the viral life cycle for which RAB1A is required
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