Supplementary MaterialsDocument S1. replication fitness of every viral mutant between mosquito and human cells, we identified that mutations affecting glycosylation display the most divergence. By characterizing individual mutants, we show that ablation of glycosylation selectively benefits ZIKV contamination of mosquito cells by enhancing cell entry, whereas it either has little impact on ZIKV contamination on certain human cells or leads to decreased contamination through the entry factor DC-SIGN. In conclusion, we define the functions of individual residues of ZIKV envelope protein, which contribute GM 6001 kinase inhibitor to ZIKV replication fitness in human and mosquito cells. family, which also includes the dengue computer virus (DENV), West Nile computer virus (WNV), Japanese encephalitis computer virus (JEV), and yellow fever computer virus (YFV) (Lindenbach, 2007). ZIKV was first isolated from the serum of a sentinel rhesus monkey in the Zika forest of Uganda in 1947 and was subsequently recovered from the mosquito in the same forest (Dick et?al., 1952). ZIKV contamination is mostly asymptomatic, but it can cause influenza-like symptoms, such as fever, headache, joint pain, and maculopapular rash (Simpson, 1964, Duffy et?al., 2009). The recent outbreak of ZIKV in the Americas has demonstrated GM 6001 kinase inhibitor the potential for ZIKV to cause more serious disease, including microcephaly, other congenital malformations, and Guillain-Barr syndrome. The ZIKV genome consists of a 10.8-kilobase single-stranded positive-sense RNA that codes for three structural proteins (capsid [C], membrane [prM/M], and envelope [E]) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). In addition, there are short UTRs on both the 5 and 3?ends of the genome (Kuno and Chang, 2007). The mature ZIKV virion is usually roughly spherical and 50?nm in diameter. It contains a nucleocapsid that is surrounded by an icosahedral shell consisting of 180 copies of both E glycoprotein and M protein anchored in GM 6001 kinase inhibitor a lipid bilayer (Sirohi et?al., 2016, Kostyuchenko et?al., 2016). The flavivirus E protein, arranged as dimers on the surface of the mature virion, is the major viral Plxdc1 protein involved in GM 6001 kinase inhibitor host-cell entry factor binding and fusion (Lindenbach, 2007). Each E protein monomer consists of four GM 6001 kinase inhibitor domainsthree ectodomains (DI, DII, and DIII) and a transmembrane domain name (TM). The structurally central DI acts as a bridge between DII and DIII and contains one N-linked glycosylation site (N154). The N-linked glycosylation at residue 153/154 of the E protein is usually conserved across most flaviviruses and has been shown to be important for optimal contamination of mosquito and mammalian cells (Lee et?al., 2010, Roehrig et?al., 2007, Post et?al., 1992, Heinz and Allison, 2003). DII includes the dimerization interface and a fusion loop that interacts with the endosomal membrane after conformation change. The IgG-like DIII is usually a continuous polypeptide segment and is thought to be important for binding to entry factors. Several host entry factors, including DC-SIGN, AXL, and TYRO3, have been shown to be important for mediating ZIKV contamination (Hamel et?al., 2015, Nowakowski et?al., 2016). However, the detailed mechanism by which the E protein interacts with host-cell entry factors or the sequence determinants that contribute to human versus mosquito cell tropisms is not fully known. We have developed a high-throughput fitness profiling approach that combines high-density mutagenesis with the power of next-generation sequencing to identify functional residues in the context of virus contamination (Qi et?al., 2014, Qi et?al., 2017, Remenyi et?al., 2014, Wu et?al., 2016). In this study, we applied this approach to systematically analyze the functional residues of ZIKV E protein during contamination of mosquito and human cells. We achieved high sensitivity in identifying residues essential for ZIKV E protein function. Surprisingly, we found that N-linked glycosylation at position N154 had differential effects on ZIKV contamination between mosquito and human cells. Ablation of this glycosylation had little impact on viral contamination of human cells (A549 and hCMEC), whereas it significantly increased contamination of mosquito cells (C6/36), most probably by enhancing ZIKV entry. Last, N154 glycosylation was found to be important for ZIKV contamination of mammalian cells through the entry factor DC-SIGN, further broadening our current knowledge concerning the glycosylation of E protein in the mammalian and invertebrate ZIKV life cycles. Results Establishing Infectious cDNA Clone of ZIKV Strain PRVABC59 To facilitate mutational analysis of the ZIKV genome, we generated a plasmid (pZ-PR) carrying ZIKV cDNA, which was generated from an early passage of PRVABC59 computer virus. The ZIKV PRVABC59 strain was isolated.