Basic patches on the E2 glycoprotein of eastern equine encephalitis virus influence viral vascular clearance and...

Previously, we found that chimeric Sindbis-eastern equine encephalitis virus (SINV-EEEV) particles can be removed from the murine blood circulation in a phagocyte-dependent manner which can be disrupted by either transient depletion of vascular heparan sulfate (HS) glycosaminoglycans (GAGs), or mutation of the viral E2 glycoprotein (K71/74/77A) associated with decreased GAG binding in vitro. Here, we further investigate the viral determinants of EEEV vascular clearance and evaluate their role in viremia development. We identified two large basic patches on the EEEV E2 glycoprotein which contain two known GAG-binding sites (K71/74/77 and K156/R157) and six additional basic residues (K10, R13, K56, R152, K231, and K232). We find that disruption of either basic patch by single alanine substitutions promotes prolonged retention of SINV-EEEV particles in the murine blood circulation in an experimental viremia model. Furthermore, we observed that the K156/R157A, K10A, and K231A mutations are also associated with similar viral dissemination in a mouse infection model as the attenuated K71/74/77A mutant. Surprisingly, despite known differences in GAG binding and potential alteration in receptor interactions, we find the initial dispersal of wild-type (WT) and mutant SINV-EEEV virions from the inoculation site to the draining lymph node to be equivalent at 1 hour post-subcutaneous inoculation. Moreover, our data suggest the higher viremia associated with mutation of the E2 basic patches may be attributed to evasion of viremic control by blood-filtering phagocytes. Overall, this study defines viral features of the EEEV E2 glycoprotein that influence tissue-specific viral dissemination and highlights the capacity of blood-filtering phagocytes to modulate EEEV viremia.IMPORTANCEVirus-GAG interactions have long been studied in vitro; however, investigating the impact of these interactions in vivo has been challenging. Previously, we showed that blood-filtering phagocytes and vascular HS mediate the removal of enhanced GAG-binding WT SINV-EEEV virions from the blood circulation in a reductionist, experimental viremia model. Here, we demonstrate that single-residue, charge-neutralizing mutations within basic patches of the E2 glycoprotein are sufficient both to promote viral evasion of vascular clearance and viral dissemination in an infection model. We also find that the WT and decreased GAG-binding SINV-EEEV virions traffic similarly from a subcutaneous inoculation until drainage into the bloodstream, upon which the WT virus is selectively depleted. These observations suggest viral dissemination is influenced by tissue-specific, virion-GAG interactions.