The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by Nlinked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential Nlinked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

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The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.