Viral attenuation due to the increased electrostatic interactions through negatively

The attenuation of virulent viruses has often been achieved by the serial passages in vitro in order to increase the interactions between the virions and the negatively charged molecules on the cellular surface. The gain of positive charges on such locations has been hypothesized to be the consequence of the adaptation made by the various viruses to the environment of non-natural hosts or tissue cultures. Although the post-translation modification of GAG has been considered as the potential underlying mechanism that mediates viral entry in vitro through the electrostatic interactions, the GAG molecules are also likely to cause the attenuation of virulent strains in the experimental models of vertebrate hosts predominantly through the retention of progeny virions after the processes of morphogenesis and virion secretion are completed. The attenuated GAG- binding variant viruses exhibit a higher affinity to heparin or heparan molecules that may be used as the approach for phenotype characterization (Klimstra, Ryman et al. 1998, Perera-Lecoin, Meertens et al. 2014).

The attenuation of YFV being attributed to the positive charges on the E protein has been reported in two experimental models. The first involves the T380R mutation that was described in the section 3.1.A and which shares a similar phenotype with the GAG-binding variants of flaviviruses (Lee and Lobigs 2008). The second involves the characterization of the K326E mutation in the neuroadapted 17D strain which implicated the BC loop as a potential locus for the binding with negatively charged cellular molecules and the neurovirulence of the virus (Nickells, Cannella et al. 2008).

The attenuation caused by the mutations in the GAG-binding region of EDIII was first observed in MVEV. The RGD motif in MVEV EDIII was found to be critical for the growth kinetics in vitro and the virulence in vivo. The conservative mutation from aspartate to glutamate resulted in the minor reduction in the viral titers and the virulence; whereas, the non-conservative glycine, histidine, and tyrosine mutations attenuated the virus and led to the lower mortality rates among the outbred Swiss mice. Interestingly, the gain of positive charge by the histidine mutation not only was responsible for the most significant attenuation by causing no or negligible mortality among intraperitoneally challenged mice, but it also increased the inhibition of viral entry caused by heparin sulfate treatment (Lee and Lobigs 2000). In the other independent study characterizing the hinge region and the RGD motif of MVEV E protein, the

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non-conservative mutation of the aspartate residue to asparagine or tyrosine significantly reduced both the neurovirulence and neuroinvasiveness in intracranially or intraperitoneally inoculated outbred Swiss mice. Whilst the growth kinetics showed that the non-conservative mutations impaired the viral replication, the pH-dependent conformational change was not altered as the optimal pH of hemagglutination activity remained the same. The lower viral titers in the brains of intracranially challenged mice further supported the attenuation caused by the mutations (Hurrelbrink and McMinn 2001). The RGD motif is therefore likely to harbor the determinant for viral attenuation by enhancing the interactions with negatively charged cellular materials.

The serial passage experiments of other flaviviruses within the JEV serocomplex in naturally nonsusceptibile tissues further identified additional locations in the E protein associated with viral attenuation. The additional three amino acid substitutions were identified as the lysine substitutions in the glutamate 49, 138, and 306 of the envelope proteins of viral variants which were shown to have higher GAG binding affinity (Lee, Hall et al. 2004). However, the results still support the importance of mutations in RGD motif of EDIII which was previously tested in the mutants generated by the reverse genetics systems for viral attenuation. The mutations still appeared in the regions corresponding to the RGD motif in the EDIIIs of KUNV MRM61C strain and JEV SA14 strain after the wildtype viruses were serially passaged in human adenocarcinoma SW13 cells. The mutation of Glu390, which shares the biochemical properties of the aspartate in the RGD motif, to glycine in KUNV EDIII has been identified in three plaque- purified variants. Experimentally, the Asp389 in the EDIII of JEV SA14 strain was substituted with glycine in one of the five JEV variants. Both of the mutations in KUNV and JEV were considered as the non-conservative mutations with the replacement of the acidic electrically charged side chains of glutamate and aspartate with the small side chain of glycine. Both variants favored the binding of GAG molecules in vitro and significantly reduced the neuroinvassiveness in the intraperitoneally challenged Swiss mice. In addition to the mutations in the FG loops of EDIII, the glutamatelysine mutation at the residue 38 of WNV E protein and JEV E protein was detected after the five serial passages in SW13 cells. Both of the mutations are located in the E0 β-strand in EDI of WNV and JEV as part of the external sheet structure of EDI exposed on

the surface of flavivirus virions (Rey, Heinz et al. 1995). The increase of the binding to the heparin was observed among the mutants in vitro and the LD50 for intracerebral and

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tested with the WNV cDNA infectious clone (Lee, Hall et al. 2004). The glutamate  lysine mutation at the residue 49 in the EDI of JEV also increased the affinity of heparin binding but only led to the partial attenuation by the reduction of mortality by 80%. The mutants of KUNV and WNV that caused the increase of heparin binding and the attenuation in mice were removed more efficiently from the blood circulation. Together, the amino acid substitutions with basic amino acid residues among encephalitic mosquito-borne flaviviruses are the consequences of the viral adaptation after the serially passage in vitro. The mutants favored the binding to the negatively charged molecules in the vertebrate hosts and led to the viral attenuation through the higher efficiency of viral clearance in the blood stream. The available one-plasmid reverse genetics system of JEV further supported the role of the lysine substitution at the residue 138 and revealed the retention of progeny virions in the infected cells, which was consistent with the proposed model of the attenuation of the GAG-binding variant of recombinant YFV described by Lee et al (Zhao, Date et al. 2005, Lee and Lobigs 2008). On the other hand, the lysine substitution at the residue 306 also occurred in variants of the T1P1 strain isolated from Armigeres subalatus and purified based on the difference in plaque morphologies and led to the delay in the peak viral titer in the brain of intracranially inoculated ICR mice confirming its potential contribution to the attenuation (Chiou and Chen 2007).

The dependence of utilizing GAG molecules for the retention of variant viruses was further tested in different strains of DENV-2, which have a different history of passage in vitro. The mouse neurovirulent New Guinea C strain acquired the lysine substitution at the residue 126 of the envelope protein and this led to the higher affinity of GAG binding, which differs from other natural isolates of DENV-2. The gain of positive charges due to the lysine substitutions at the residue 120 and the arginine substitutions at the residue 227 led to the attenuation in AG129 mice; whereas the lysine substitution at the residue 202 resulted in the phenotypic instability without the attenuation. Also, the compensatory mutation at asparagine 124 is required for the stabilization of the structure (Lee, Wright et al. 2006). Although the gain of positive charges in DENV-2 E proteins also led to the attenuation, the locations of the mutations were found in the surface of EDII and differ from the flaviviruses in the JEV serocomplex.

Although TBEV is listed under the other distinct category of tick-borne flaviviruses and predominantly utilizes ticks as its vectors, its attenuation in vivo was associated with the phenotype of increased GAG binding after the serial passage in vitro (Mandl, Kroschewski et al.

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2001, Kroschewski, Allison et al. 2003). The adaptation of TBEV by serial passage in BHK-21 cells resulted in the substitutions with predominantly positively charged basic amino acids on the upper and lateral surface of virions across the three domains of the E protein. Three mutants, E201K, E122G and S158R-G159R, exhibited the higher sensitivity of soluble heparin inhibition and shared the phenotype of the reduced infectivity and neuroinvasiveness in Swiss mice model.

The evidence has suggested that the tropism and disease pathogenesis characteristic of different flaviviruses are likely altered due to the mutations causing the different GAG-binding properties. In addition to the phenotypic changes in the binding affinity with the GAG molecules and virulence associated with the mutations in the FG loop of the flavivirus EDIII, the structural and sequence homology between the E2 glycoprotein of alphaviruses and the E protein of flaviviruses suggested the presence of a secondary structure similar to the FG loop of flaviviruses in the domain A of the E2 glycoprotein of alphaviruses. Such structure consists of the linear polypeptide between the E and F β-stands of alphavirus E2 glycoprotein (Pierro, Powers et al. 2008, Voss, Vaney et al. 2010). The same region has been found to harbor the glutamatelysine substitution in the epizootic strains of VEEV IE subtype and associated with the GAG-binding affinity (Brault, Powers et al. 2002, Wang, Brault et al. 2003). However, there has not been any clear association identified between the enzootic and epizootic types of VEEV that utilize different vector species.

Together, the gain of positive charges on the E protein of flaviviruses has led to the attenuation in mice. Although the mutations are found in various locations clustered in the serocomplex-specific manner, the consequences of serial passage in vitro is likely to lead to the lysine or arginine substitutions that increase the GAG- binding affinity, which in turn results in the rapid viral clearance from the blood circulation. Similar negatively charged homologs are also synthesized by the mosquitoes as the post-translational modifications of cellular proteins (Kato, Dasgupta et al. 2002, Kato, Mueller et al. 2005). The evaluation of the GAG-binding variants of flaviviruses in its infectivity and disseminability in the vector mosquitoes will provide the mechanistic understanding in the role of such a conserved class of molecules in the vertebrate hosts and insect vectors in determining the vector competence for arboviruses.

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3.2 Results