Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure

The flaviviruses of major medical importance in South American countries are yellow fever, dengue, Saint Louis encephalitis, West Nile and Rocio viruses. Rocio virus (ROCV) has been responsible for epidemics of severe encephalitis in Brazil with a case-fatality rate of 10 % and development of sequelae in 20 % of the survivors. We have sequenced and characterized the entire genome of ROCV for the first time, by determining the general traits of the open reading frame and the characteristics of viral genes including the potential cleavage sites, conserved or unique motifs, cysteine residues and potential glycosylation sites. The conserved sequences in the 39-non-coding region were identified, and the predicted secondary structures during cyclization between 59-and 39-non-coding regions were studied. Multiple protein and phylogenetic analyses based on antigenically important and phylogenetically informative genes confirmed a close relationship between ROCV and Ilheus virus (ILHV), together constituting a unique and distinct phylogenetic subgroup as well as the genetic relationship of ROCV with several members of the Japanese encephalitis group. Although ROCV is phylogenetically related to ILHV, our study shows that it is still a virus distinct from the latter virus. This is the first flavivirus uniquely indigenous to Brazil that has been sequenced completely and the genome characterized. The data should be useful for further studies at the molecular level, including construction of infectious clone, identification of gene function, improved disease surveillance based on molecular diagnostic tools and vaccine development. INTRODUCTIONIn South America, until the mid-1970s, the two neurotropic flaviviruses had been Saint Louis encephalitis virus (SLEV) and Ilheus virus (ILHV). However, in contrast to North America, an epidemic of encephalitis in humans by SLEV in South America was not recorded for many years until the first outbreak in Argentina in 2005(Diaz et al., 2006, despite accumulated records of virus isolation from mosquitoes and vertebrate hosts throughout the continent (Spence, 1980). As for the other virus, ILHV, human infection was sporadic and development of neurotropic manifestations has been even more infrequent. Thus, the sudden emergence of an outbreak of encephalitis caused by a flavivirus in 1974-75 in Brazil was unexpected.Rocio virus (ROCV) belongs to the genus Flavivirus in the family Flaviviridae. Its prototype (strain SPH 34675) was isolated in Ribeira Valley in the south-eastern São Paulo state of Brazil in 1975 from the cerebellum of a fatal human case during an epidemic of encephalitis that could not be readily diagnosed with the reagents then available for known encephalitic viruses. The epidemic, which began in 1974, spread to more than 20 municipalities over the following 2 years causing approximately 1000 diagnosed cases.Members of the genus Flavivirus (hereafter called flaviviruses) are positive-sense, single-stranded RNA viruses that infect humans and other vertebrates. They are mainly...

Section: Discussionmentioning

confidence: 99%

Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in São Paulo state

Medeiros

Nunes

Vasconcelos

et al. 2007

“…However, the E protein of African strains of YF virus is not glycosylated (Ballinger-Crabtree & Miller, 1990), indicating that factors such as membrane lipid composition may affect E protein structure. TBE virus E protein has a single utilized glycosylation site, but enzymatic deglycosylation after synthesis failed to alter antigenic expression (Winkler et al, 1987), indicating that carbohydrate was not required for maintenance of the antigenic structure. On the other hand, Guirakhoo et al (1989) reported that carbohydrates stabilize TBE E protein antigens against denaturing agents.…”

Section: Discussionmentioning

confidence: 99%

Molecular and biological characterization of a non-glycosylated isolate of St Louis encephalitis virus

Vorndam¹,

Mathews

Barrett

et al. 1993

The glycosylation patterns of the envelope (E) glycoprotein of several naturally occurring strains of St Louis encephalitis (SLE) virus were investigated. SLE viruses were found that contained both glycosylated and non-glycosylated E proteins, and one isolate (Tr 9464) that lacks N-linked glycosylation sites on its E protein was identified. SLE virus monoclonal antibodies that define E protein B cell epitopes and demonstrate biological activities reacted essentially to the same extent with glycosylated and non-glycosylated virions. These results indicate that glycosylation is not essential for epitope conformation or recognition. However, failure to glycosylate the E protein was associated with possible morphogenetic differences as manifested by reduced virus yields and differences in specific infectivity.

“…This statement does not at first appear justified by the literature. Reports have demonstrated that aspects of the antigenic profile of TBEV E protein are quite stable when Asn-linked carbohydrates are removed (Winkler et al, 1987) and that removal of glycosylation may enhance JEV E immunogenicity (Zhang et al, 2011). However, E glycosylation still appears to be important to generate an authentic immune response against virulent WNV in horses, as the carbohydrate modification has been demonstrated to be crucial for the correct presentation of an antibody-inducing epitope (HobsonPeters et al, 2008).…”

Section: Glycan Modificationmentioning

confidence: 99%

“…Flaviviruses secrete three discrete glycoproteins from infected cells: prM (Calvert et al, 2012; Goto et al, 2005; Kim et al, 2008), E (Beasley et al, 2005 Goto et al, 2005;Hanna et al, 2005;Lee et al, 2010;Li et al, 2006;Mondotte et al, 2007;Scherret et al, 2001;Setoh et al, 2012;Winkler et al, 1987) and NS1 (Muller & Young, 2012;Muylaert et al, 1996; Somnuke et al, 2011). Asnlinked carbohydrate modifications are always present and are relatively conserved in prM and NS1 with one to three modifications in the pr portion of prM (believed to be important in protecting the fusion peptide in E during virion egress) (Goto et al, 2005;Hanna et al, 2005; Kim et al, 2008;Li et al, 2008;Mandl et al, 1988; von Lindern et al, 2006), and modification at two to three residues in NS1 (important for multimerization of soluble NS1) (Muller & Young, 2012;Muylaert et al, 1996; Somnuke et al, 2011).…”

Section: Glycan Modificationmentioning

confidence: 99%

Post-translational regulation and modifications of flavivirus structural proteins

Roby

Setoh

Hall

et al. 2015

102

Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular -capsid (C), pre-membrane (prM) and envelope (E) -are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics. FlavivirusesThe genus Flavivirus in the family Flaviviridae comprises small, enveloped viruses with non-segmented genomes consisting of single-stranded, positive-sense RNA. These RNA genomes are flanked by 59 and 39 untranslated regions (UTRs) and encode a single polyprotein that is cleaved coand post-translationally to generate between 10 and 11 viral proteins. The 59 UTR contains a type 1 m 7 GpppNm cap structure and the 39 UTR lacks a polyadenylated tail. Structural elements within the UTRs regulate genome replication, translation and host immune responses. Viral proteins are divided between structural proteins, which form the virion, and non-structural proteins, which are involved in genomic replication and modulating host immunity (Fig. 1a) (Lindenbach et al., 2007;Roby et al., 2012).Flaviviruses are maintained predominantly in natural transmission cycles between vertebrate reservoir species (normally mammalian or avian) and haematophagous arthropod vectors (mosquitoes or ticks). Notable exceptions are the viruses that are maintained solely in mosquito hosts (insectspecific flaviviruses) and viruses with no known invertebrate vector (Cook et al., 2012;Mackenzie et al., 2004). As of 2013, the International Committee on Taxonomy of Viruses has classified 53 different viral species as belonging to ...