Gregory D. Gromowski

Envelope Protein Glycosylation Status Influences Mouse Neuroinvasion Phenotype of Genetic Lineage 1 West Nile Virus Strains

ABSTRACT The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

ABSTRACT Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

Characterization of Dengue Virus Complex-Specific Neutralizing Epitopes on Envelope Protein Domain III of Dengue 2 Virus

ABSTRACT The surface of the mature dengue virus (DENV) particle is covered with 180 envelope (E) proteins arranged as homodimers that lie relatively flat on the virion surface. Each monomer consists of three domains (ED1, ED2, and ED3), of which ED3 contains the critical neutralization determinant(s). In this study, a large panel of DENV-2 recombinant ED3 mutant proteins was used to physically and biologically map the epitopes of five DENV complex-specific monoclonal antibodies (MAbs). All five MAbs recognized a single antigenic site that includes residues K310, I312, P332, L389, and W391. The DENV complex antigenic site was located on an upper lateral surface of ED3 that was distinct but overlapped with a previously described DENV-2 type-specific antigenic site on ED3. The DENV complex-specific MAbs required significantly higher occupancy levels of available ED3 binding sites on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Additionally, there was a great deal of variability in the neutralization efficacy of the DENV complex-specific MAbs with representative strains of the four DENVs. Overall, the differences in physical binding and potency of neutralization observed between DENV complex- and type-specific MAbs in this study demonstrate the critical role of the DENV type-specific antibodies in the neutralization of virus infectivity.

Characterization of dengue complex-reactive epitopes on dengue 3 virus envelope protein domain III

The disease dengue (DEN) is caused by four genetically and serologically related viruses termed DENV-1, -2, -3, and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3. The ED3 contains the DENV type-specific and DENV complex-reactive (epitopes shared by DENV 1-4) antigenic sites. In this study the epitopes recognized by four DENV complex-reactive monoclonal antibodies (MAbs) with neutralizing activity were mapped on the DENV-3 ED3 using a combination of physical and biological techniques. Amino acid residues L306, K308, G381, I387, and W389 were critical for all four MAbs, with residues V305, E309, V310, K325, D382, A384, K386, and R391 being critical for various subsets of the MAbs. A previous study by our group (Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue complex-specific neutralizing epitopes on the envelope protein domain III of dengue 2 virus. J. Virol 82, 8828-8837) characterized the same panel of MAbs with DENV-2. The location of the DENV complex-reactive antigenic site on the DENV-2 and DENV-3 ED3s is similar; however, the critical residues for binding are not identical. Overall, this indicates that the DENV complex-reactive antigenic site on ED3 may be similar in location, but the surprising result is that DENV 2 and 3 exhibit unique sets of residues defining the energetics of interaction to the same panel of MAbs. These results imply that the amino acid sequences of DENV define a unique interaction network among these residues in spite of the fact that all flavivirus ED3s to date assume the same structural fold.

Mutations of an antibody binding energy hot spot on domain III of the dengue 2 envelope glycoprotein exploited for neutralization escape

Previous crystallographic studies have identified a total of 11 DENV-2 envelope protein domain III (ED3) residues (K305, F306, K307, V308, V309, K310, I312, Q325, P364, K388, and N390) that interacted, through both side- and main-chain contacts, with the Fab of a dengue virus (DENV) subcomplex-specific neutralizing monoclonal antibody (MAb) 1A1D-2 (Lok et al., 2008). Here, we used DENV-2 recombinant ED3 mutants of the MAb 1A1D-2 structural epitope residues to determine the functional epitope of this MAb. The side-chains of residues K307, K310 and I312 were determined to be functionally critical for MAb binding, and thus constitute a hot spot of binding energy for MAb 1A1D-2 on the DENV-2 ED3. Overall, these findings demonstrate that only a subset of the amino acid residue side-chains within the structural epitope of MAb 1A1D-2 define a functional epitope on the DENV-2 ED3 that is essential for MAb binding and neutralization escape.

Venezuelan Equine Encephalitis Virus Transmission and Effect on Pathogenesis

Quantifying the dose of an arbovirus transmitted by mosquitoes is essential for designing pathogenesis studies simulating natural infection of vertebrates. Titration of saliva collected in vitro from infected mosquitoes may not accurately estimate titers transmitted during blood feeding, and infection by needle injection may affect vertebrate pathogenesis. We compared the amount of Venezuelan equine encephalitis virus collected from the saliva of Aedes taeniorhynchus to the amount injected into a mouse during blood feeding. Less virus was transmitted by mosquitoes in vivo (geometric mean 11 PFU) than was found for comparable times of salivation in vitro (mean saliva titer 74 PFU). We also observed slightly lower early and late viremia titers in mice that were needle injected with 8 PFU, which represents the low end of the in vivo transmission range. No differences in survival were detected, regardless of the dose or infection route.

Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III

Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.

Characterization of a dengue type-specific epitope on dengue 3 virus envelope protein domain III

Dengue virus (DENV) is a mosquito-borne disease caused by four genetically and serologically related viruses termed DENV-1, -2, -3 and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2 and ED3. The ED3 domain contains DENV type-specific and DENV complex-reactive antigenic sites. To date, nearly all antigenic studies on the E protein have focused on DENV-2. In this study, the epitope recognized by a DENV-3 type-specific monoclonal antibody (mAb 14A4-8) was mapped to the DENV-3 ED3 domain using a combination of physical and biological techniques. Epitope mapping revealed that amino acid residues V305, L306, K308, E309, V310, K325, A329, G381 and I387 were critical for the binding of mAb 14A4-8 and amino acid residues T303, K307, K386, W389 and R391 were peripheral residues for this epitope. The location of the mAb 14A4-8 epitope overlaps with the DENV complex-reactive antigenic site in the DENV-3 ED3 domain.

Conservation of the DENV-2 type-specific and DEN complex-reactive antigenic sites among DENV-2 genotypes

The envelope (E) protein is composed of three domains (ED1, ED2 and ED3) with ED3 targeted by the most potent neutralizing antibodies. DENV-2 strains can be divided into six genotypes. Comparison of ED3 of representative strains of the six genotypes revealed that there are nine variable residues that are specific to a given genotype. Recombinant ED3s (rED3s) of six different DENV-2 strains representing all nine variable residues were expressed, and their reactivity against a panel of two DENV-2 type-specific and three DENV complex-reactive monoclonal antibodies (mAbs) were compared. The differences in binding affinity to the rED3s representing different DENV-2 genotypes were relatively small, with the exception of type-specific-mAb 3H5 that showed up to 10-fold differences in binding between genotypes. Overall the binding differences did not lead to detectable differences in neutralization. Based on these results, DENV-2 ED3-specific neutralizing antibodies will likely be effective against DENV-2 strains from all six genotypes.

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues

The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants.