Comparative genomic analysis of pre-epidemic and epidemic Zika virus strains for virological factors potentially associated with the rapidly expanding epidemic

Zhu, Zheng; Chan, Jasper Fuk-Woo; Tee, Kah-Meng; Choi, Garnet Kwan-Yue; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Tse, Herman; Yuen, Kwok-Yung

Our genomic analysis revealed some changes in the 3ʹ-UTR sequence of the post-2007 epidemic ZIKV strain. The 5ʹ and 3ʹ terminal sequences of the genome of flaviviruses fold into conserved RNA secondary structures and encode regions essential for genome cyclization at the initial phase of replication. 24 In mosquito-borne flaviviruses including ZIKV, the 3ʹ UTR is further divided into three domains, including the highly variable proximal domain 1 that directly follows the stop codon, the moderately conserved domain 2 that contains the SL and DB structures, and the highly conserved domain 3 that contains the complementary cyclization elements and the conserved sHP-3ʹ SL structure. Deletion of the SL sequences in the 5ʹ-or 3ʹ-UTR is lethal for flavivirus infectious clones. 25, 26 These secondary RNA structures bind to host proteins, such as elongation factor 1α and poly(A)-binding protein, and proteins of the viral replication complex, including C, NS2A, NS3 and NS5 proteins, to promote genomic RNA cyclization. 27 Genomic RNA cyclization is essential for viral replication in two ways. First, the 5ʹ-SLA acts as a promotor element to stimulate the NS5 RdRp to initiate negative strand synthesis at the 3ʹ-UTR. [28] [29] [30] Second, the 5ʹ-and 3ʹ-UTRs move into close proximity for cap-dependent translation of the viral polyprotein to proceed. [31] [32] [33] The two most conserved secondary RNA structures in flavivirus genomes are the Y-shape SLA structure at the 5ʹ-UTR and the sHP-3ʹ SL structure at the 3ʹ-UTR. 24 Expectedly, these are also present in the ZIKV genomes. The arrangement and sequences of the other 3ʹ-UTR RNA secondary structures of ZIKV are less conserved from those of other flaviviruses. 24 For example, there are two DB structures and two SL structures in addition to the conserved sHP-3ʹ SL structure in DENV-1 and DENV-3, whereas three additional putative SL and one DB structures are found in both the pre-epidemic and epidemic ZIKV strains. 24 Interestingly, we found a large bulge of nine nucleotide bases at the SLI of the epidemic ZIKV strain, which more closely resembles the SLII than the corresponding SLI of the pre-epidemic After cleavage of the polyprotein, the C protein of flaviviruses is released into the cytoplasm and forms homodimers. The basic residues on one side of the C protein bind the RNA genome and the hydrophobic residues on the other side interact with the viral lipid envelope. 34, 35 Even after virus-endosomal membrane fusion, the entering viral genome may remain associated with the C dimers to evade from host nucleases and RNA sensors. Thus, the C protein of flaviviruses may function as an RNA chaperone in addition to its role in the formation of viral nucleocapsid. The resulting nucleocapsid then buds into the endoplasmic reticular lumen to form viral particles with the prM and E proteins. 36, 37 The C protein may also be found in the nuclei and nucleoli of cell lines infected by flaviviruses, including DENV-2, DENV-4, WNV, JEV and Kunjin virus. [38] [39] [40] The migration of the C protein to the nuclei and nucleoli are believed to be mediated by nuclear localization signals. 41 In ZIKV, we found one putative nuclear localization signal near the 3ʹ-end of the C protein, which is conserved in the pre-epidemic and epidemic strains (Figure 2A ). JEV with a single point mutation (T45G) at the N terminus of the C coding region has reduced virulence. 42 In our study, we found five amino acid substitutions in C pr