The absence of secondary infection by the H244A DENV1 mutant could be due to decreased virus particle production and/or production of particles that are non-infectious. Efficient DENV particle production is dependent on E protein folding, particle budding into the ER, and subsequent particle egress through the secretory pathway. To investigate these issues, we took advantage of the ability of the flavivirus prM and E proteins to assemble into virus-like particles (VLP) in the absence of other viral components or virus infection [44, 45, 46 ]. The VLP system avoids complications arising from selection of revertants of deleterious virus mutations such as H244A. Flavivirus VLP bud into the ER in the immature prM form, undergo furin maturation during transport through the secretory pathway, and display similar low pH- [34] , potential key residues in pr-E interaction are indicated by their numbers in the DENV2 NGC proteins. B) H244A mutation inhibits pr-E binding in pull-down assay. WT or H244A mutant forms of DI/II were assayed for binding to pr-sepharose beads as in Fig. 2A . C) H244A mutation inhibits pr-E binding in SPR assay. WT or H244A mutant forms of DI/II were assayed for binding to pr at various pH values using SPR as in Fig. 2C , shifting to buffer alone at 300 s. Where indicated, mAb 4G2 (molar ratio 1:1) was pre-incubated 15 min at room temperature with DI/II proteins at pH 6.0 prior to assay. Data are a representative example of two independent experiments. doi:10.1371/journal.ppat.1001157.g005 dependent fusion activity as infectious virions [44, 47] . The VLP system has been used extensively to follow the process of flavivirus particle production and the role of prM in this process [37, 44, 45, 48] .