Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes.

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.

Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein.

The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK(2) cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication.