The role of heparan sulfate in poxvirus infections

Abstract

The heparan sulfate (HS) component of heparan sulfate proteoglycans (HSPGs) has been implicated in the initiation of several viral infections, including vaccinia virus (VACV). A cell infected with VACV releases two different forms of VACV, namely the mature virus (MV) released following the death of infected cells and which infects neighbouring cells, and the enveloped virus (EV) ejected from infected cells for long-range dissemination. The relative role of HS in the infectivity of the different forms of VACV is unclear. Furthermore, there is little known about the fine specificity of the VACV-HS interactions. Therefore, in order to develop HS based molecules that could potentially have antiviral properties against HS-dependent viral infection, VACV was used as a prototype virus to understand the structural and functional consequences of the interaction between VACV and HS. ELISA studies described in Chapter 3 were used to evaluate the specificity of the MV form of VACV for heparin, differentially sulfated HS, chondroitin sulfate (CS) A-D and hyaluronic acid (HA). Lack of appropriate EV specific antibodies meant that similar ELISA studies could not be performed for the EV form of VACV. Nevertheless, the MV form of VACV bound to immobilized heparin and highly sulfated HS (HShi) with high avidity, compared to lowly sulfated HS (HSlow). The MV particles also bound to CS A-D, however, very weakly. Furthermore, the ability of the MV rich Western Reserve (WR) strain of VACV to form plaques in vitro was affected by soluble heparin, WR plaque numbers being reduced 5-fold with an incremental increase in plaque size. The formation of plaques by the EV rich International Health Department-J (IHD-J) strain was also affected in the presence of heparin, there being a 10-fold reduction in plaque numbers, an incremental increase in plaque size and the disappearance of the trademark ‘comet’ shaped plaques. These data suggest that HS recognition plays a significant role in both MV and EV infectivity, with this recognition being more important for EV infectivity. To better understand the interaction between heparin/HS and the two forms of VACV, green fluorescent protein (GFP) expressing recombinant strains of VACV were constructed, as described in Chapter 4. Subsequent inhibition of infectivity assays, performed using soluble glycosaminoglycans (GAGs), suggested that sulfated GAGs more easily inhibited EV infections than the MV infections, with heparin and HShi being highly potent inhibitors of infection. Furthermore, the ability of the EV form of VACV to establish an infection was significantly reduced in cells treated with the HS-degrading enzyme heparanase and in cells genetically deficient in HS production, compared to the MV form of VACV which appeared largely unaffected. These findings confirmed that recognition of cell surface HS is vital for EV infectivity but less important for the infectivity of the MV form of VACV. In Chapter 5, the ability of soluble heparin/HS molecules and HS mimetics…