Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles

The Yellow fever virus (YF) belongs to the genus Flavivirus within the family Flaviviridae. Members of the Flavivirus genus are typically transmitted to vertebrates by mosquitoes or ticks and frequently cause significant human morbidity and mortality (reviewed in reference 25). Human pathogens include dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, and YF. The estimated 100 million cases of dengue virus infection per year worldwide (5) and the emergence and spread of West Nile virus in the Eastern United States underscore the need for continued efforts to develop effective and inexpensive flavivirus vaccines. For YF, a live attenuated vaccine strain (17D) has been used effectively for almost 65 years. However, YF remains an enduring global public health problem due to the endemic persistence of mosquito-borne disease in sub-Saharan Africa and South America and recent reports of six fatalities temporally associated with live-virus vaccination (22, 33). The YF genome is a positive-sense RNA approximately 11 kb in length that is capped at the 5′ end but lacks a 3′ poly(A) tract. The RNA contains a single large open reading frame that is cleaved co- and posttranslationally by host cell and viral proteases (reviewed in reference 16). The polyprotein is arranged with the structural proteins at the amino terminus (C-prM-E), followed by the nonstructural (NS) proteins (NS1 through NS5). The arrangement of the proteins is NH2-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-COOH. Most of the cleavages releasing the structural proteins are mediated by host cell signal peptidase. Exceptions include prM cleavage into pr and M by the host cell enzyme furin shortly before virus release and the cleavage generating the C terminus of the virion C protein by the virus-encoded serine protease (NS2B-3 protease). This serine protease, consisting of NS2B and the N-terminal part of NS3, produces the N termini of NS2B, NS3, NS4A, 2K, and NS5. The enzyme responsible for cleavage at the NS1/2A site is unknown. Although all flavivirus proteins stem from a single polyprotein, the structural and NS proteins have been viewed as functionally distinct modules responsible for virion formation and RNA replication, respectively. For example, coexpression of prM and E is sufficient for secretion of subviral particles that mimic the subunit structure and fusogenic capabilities of the mature virion envelope (2, 29). Subgenomic replicons lacking the structural proteins replicate efficiently and can be packaged by trans expression of the structural proteins (11). Although initially thought to be involved in virion morphogenesis or release, secreted glycoprotein NS1 plays an essential role in RNA replication (18). These observations have reinforced a modular view of the flavivirus polyprotein with the dividing line between structural proteins and replicase drawn at the E/NS1 junction. The only known exception is the NS2B-3 serine protease-mediated cleavage at the C terminus of mature C, a cleavage that is a necessary prerequisite for signalase generation of the prM N terminus and virus production (4). Specific functions have been attributed to many flavivirus proteins (reviewed in reference 16), but little is known about the function of NS2A. NS2A is a small hydrophobic protein of about 22 kDa (8). Consistent with a role in RNA replication, studies with Kunjin virus (KUN) have shown that NS2A colocalizes with double-stranded RNA in discrete cytoplasmic foci and interacts with the 3′ untranslated region of KUN RNA, as well as NS3 and NS5 (20). NS2A has also been implicated in the Japanese encephalitis virus-induced cytopathic effect (CPE) (10). In YF-infected cells, 22- and 20-kDa forms of NS2A have been identified and both of these forms possess the same N-terminal sequence (8). The 20-kDa form (called NS2Aα) is believed to result from an additional internal cleavage by the NS2B-3 serine protease. Cleavage by the YF serine protease occurs at a consensus sequence consisting of two basic amino acids followed by an amino acid with a short side chain (RR↓S/G) (6). However, in the case of the NS4A/2K junction, the cleavage site is QR↓S (14). Based on the known cleavage sites, as well as substitutions at these sites that are tolerated (9, 15, 26), NS2A residues 189 to 191 (QK↓T) were identified as a possible target for the viral serine protease. Consistent with this hypothesis, replacement of Lys-190 with Ser resulted in loss of the 20-kDa protein (26). Inhibition of cleavage at the NS2A/2B site did not abrogate production of NS2Aα, indicating that processing at the NS2A/2B site was not a prerequisite for cleavage at the NS2Aα site (26). It was further shown that the NS2A Lys-190-Ser mutation blocked production of infectious virus (26). Although the roles of NS2A and this additional cleavage in YF replication were unknown, a block at the level of YF RNA replication seemed most likely. In this report, we show that NS2Aα cleavage site mutants have unimpaired RNA replication but are unable to produce infectious virus. This defect in virus production can be complemented by NS2A or NS2Aα supplied in trans and compensated for by mutations at the NS2Aα cleavage site or by second-site changes in the helicase domain of NS3. These results reveal a more complex interplay between the NS proteins and virus production than previously suspected.