Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells.

BACKGROUND: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication. RESULTS: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection. CONCLUSIONS: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.

Tissue-specific expansion of Zika virus isogenic variants drive disease pathogenesis.

BACKGROUND: The Asian lineage Zika virus (ZIKV) emerged as a public health emergency in 2016 causing severe neurological pathologies with no apparent historical correlate to the mild, disease-causing innocuous member of the mosquito-borne flavivirus genus that was discovered in Africa in 1947. Replication error rate of RNA viruses combined with viral protein/RNA structural plasticity can lead to evolution of virus-induced pathogenicity that is critical to identify and validate. METHODS: Infection studies in cells and A129 interferon alpha/beta receptor deficient mice with ZIKV French Polynesian H/PF/2013 clinical isolate, plaque-purified isogenic clone derivatives as well as infectious cDNA clone derived wild-type and site-specific mutant viruses, were employed together with Next-Generation Sequencing (NGS) to pin-point the contributions of specific viral variants in neurovirulence recapitulated in our ZIKV mouse model. FINDINGS: NGS analysis of the low-passage inoculum virus as well as mouse serum, brain and testis derived virus, revealed specific enrichment in the mouse brain that were not found in the other tissues. Specifically, non-structural (NS) protein 2A variant at position 117 along with changes in NS1 and NS4B were uniquely associated with the mouse brain isolate. Mutational analysis of these variants in cDNA infectious clones identified the NS2A A117V as the lethal pathogenic determinant with potential epistatic contribution of NS1 and NS4B variants in ZIKV brain penetrance. INTERPRETATION: Our findings confirm that viral subpopulations drive ZIKV neuropathogenicity and identify specific sequence variants that expand in the mouse brain that associates with this phenotype which can serve as predictors of severe epidemics. FUNDING: Duke-NUS Khoo Post-doctoral Fellowship Award 2020 (KWKC) and National Medical Research Council of Singapore grants MOH-000524 (OFIRG) (SW) and MOH-OFIRG20nov-0002 (SGV).

Therapeutics for flaviviral infections.

Flaviviruses are vector-borne pathogens capable of causing devastating human diseases. The re-emergence of Zika in 2016 notoriously led to a widescale epidemic in the Americas. New daunting evidence suggests that a single mutation in Zika virus genome may increase transmission and pathogenesis, further highlighting the need to be prepared for flavivirus outbreaks. Dengue, in particular infects about 400 million people each year, leading to reoccurring local outbreaks. Public health efforts to mitigate flavivirus transmission is largely dependent on vector control strategies, as only a limited number of flavivirus vaccines have been developed thus far. There are currently no commercially available antivirals for flaviviruses, leaving supportive care as the primary treatment option. In this review, we will briefly paint a broad picture of the flavivirus landscape in terms of therapeutics, with particular focus on viral targets, promising novel compounds entering the drug discovery pipeline, as well as model systems for evaluating drug efficacy.

Intra-Host Diversity of Dengue Virus in Mosquito Vectors.

Dengue virus (DENV) is the most common arbovirus, causing a significant burden on both the economy and global healthcare systems. The virus is transmitted by Aedes species of mosquitoes as a swarm of closely related virus genomes, collectively referred to as a quasispecies. The level of genomic diversity within this quasispecies varies as DENV moves through various ecological niches within its transmission cycle. Here, the factors that influence the level of DENV quasispecies diversity during the course of infection in the mosquito vectors are reviewed.

Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication.

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 310 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations. Using reverse genetics system, we either introduced rigidity in the linker through mutation to a proline or flexibility through a glycine mutation at position 266. ZIKV NS5 Val 266 to Pro mutation was lethal for viral RNA replication while the Gly mutation was severely attenuated. Serial passaging of cell culture supernatant derived from C6/36 mosquito cells transfected with mutant ZIKV RNA showed that the attenuation can be rescued. Next generation deep sequencing revealed four single nucleotide polymorphisms that occur with an allele frequency >98%. The single non-synonymous NS5 mutation Glu419 to Lys is adjacent to RdRp motif G at the tip of the fingers subdomain, while the remaining three are synonymous variants at nucleotide positions 1403, 4403 and 6653 in the genome. Reverse engineering the changes into the ZIKV NS5/Val266Gly background followed by serial passaging revealed that residue 266 is under strong positive selection to revert back to Val. The interaction of the specific conformation of the NS5 linker with Val at position 266 and the RNA binding motif G region may present a potential strategy for allosteric antiviral drug development.

Attenuated dengue viruses are genetically more diverse than their respective wild-type parents.

Dengue poses a significant burden of individual health, health systems and the economy in dengue endemic regions. As such, dengue vaccine development has been an active area of research. Previous studies selected attenuated vaccine candidates based on plaque size. However, these candidates led to mixed safety outcome in clinical trials, suggesting it is insufficiently informative as an indicator of dengue virus (DENV) attenuation. In this study, we examined the genome diversity of wild-type DENVs and their attenuated derivatives developed by Mahidol University and tested in phase 1 clinical trials. We found that the attenuated DENVs, in particular the strain under clinical development by Takeda Vaccines, DENV2 PDK53, showed significantly higher genome diversity than its wild-type parent, DENV2 16681. The determinant of genomic diversity was intrinsic to the PDK53 genome as infectious clone of PDK53 showed greater genomic diversity after a single in vitro passage compared to 16681 infectious clone. Similar trends were observed with attenuated DENV1 and DENV4, both of which were shown to be attenuated clinically, but not DENV3 that was not adequately attenuated clinically. Taken together, evidence presented here suggests that genome diversity could be developed into a marker of DENV attenuation.

Vaccination and Therapeutics: Responding to the Changing Epidemiology of Yellow Fever.

At the turn of the nineteenth century, yellow fever (YF) was considered the most dangerous infectious disease with high case fatality. Subsequent, mass vaccination campaigns coupled with widespread elimination of the YF mosquito vector significantly decreased YF cases and reduced outbreaks to the tropical and subtropical forested regions of Africa and South America. However, recent (2016) large outbreaks in Angola, Democratic Republic of Congo (DRC), and South-Eastern Brazil, where previously had been demarcated as low-risk regions, have highlighted the possibility of a rapidly changing epidemiology and the potential re-emergence of yellow fever virus (YFV). Furthermore, the first-ever importation of YFV into Asia has highlighted the potential fear of YFV emerging as a global threat. In this review, we describe the changing epidemiology of YF outbreaks, and highlight the use of public health policies, therapeutics, and vaccination as tools to help eliminate future YFV outbreaks.

Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein.

The SARS-CoV-2 outbreak originated in China in late 2019 and has since spread to pandemic proportions. Diagnostics, therapeutics and vaccines are urgently needed. We model the trimeric Spike protein, including flexible loops and all N-glycosylation sites, in order to elucidate accessible epitopes for antibody-based diagnostics, therapeutics and vaccine development. Based on published experimental data, six homogeneous glycosylation patterns and two heterogeneous ones were used for the analysis. The glycan chains alter the accessible surface areas on the S-protein, impeding antibody-antigen recognition. In presence of glycan, epitopes on the S1 subunit, that notably contains the receptor binding domain, remain mostly accessible to antibodies while those present on the S2 subunit are predominantly inaccessible. We identify 28 B-cell epitopes in the Spike structure and group them as non-affected by the glycan cloud versus those which are strongly masked by the glycan cloud, resulting in a list of favourable epitopes as targets for vaccine development, antibody-based therapy and diagnostics.

Vaccination and Therapeutics: Responding to the Changing Epidemiology of Yellow Fever.

PURPOSE OF REVIEW: At the turn of the nineteenth century, yellow fever (YF) was considered the most dangerous infectious disease with high case fatality. Subsequent, mass vaccination campaigns coupled with widespread elimination of the YF mosquito vector significantly decreased YF cases and reduced outbreaks to the tropical and subtropical forested regions of Africa and South America. RECENT FINDINGS: However, recent (2016) large outbreaks in Angola, Democratic Republic of Congo (DRC), and South-Eastern Brazil, where previously had been demarcated as low-risk regions, have highlighted the possibility of a rapidly changing epidemiology and the potential re-emergence of yellow fever virus (YFV). Furthermore, the first-ever importation of YFV into Asia has highlighted the potential fear of YFV emerging as a global threat. SUMMARY: In this review, we describe the changing epidemiology of YF outbreaks and highlight the use of public health policies, therapeutics, and vaccination as tools to help eliminate future YFV outbreaks.