Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

Teramoto, Tadahisa; Boonyasuppayakorn, Siwaporn; Handley, Misty D.; Choi, Kyung Hyun; Padmanabhan, Radhakrishnan

doi:10.1074/jbc.m114.584466

Cited by 20 publications

(32 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Viral RNAs from supernatants of infected C6/36 cells were extracted using a ZR viral RNA kit (Zymo Research) according to the manufacturer's instructions. Synthesis of cDNAs by reverse transcriptase (RT) were performed by incubation of RNAs with Moloney murine leukemia virus (M-MuLV) RT, random primers, and deoxynucleoside triphosphates (dNTPs) (New England Biolabs) at 42°C for 2 h. To quantify DENV2 RNA copy number, quantitative RT-PCR (qRT-PCR) was performed in duplicate using iTaq Universal SYBR green Supermix (Bio-Rad), as described previously (82). The average threshold cycle (C T ) values were converted to viral copy numbers by comparing with the standard plot established from known amounts of DENV2 cDNA.…”

Section: Cell Culture and Virus Infectionsmentioning

confidence: 99%

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

Teramoto

Huang

Armbruster

et al. 2019

J Virol

Self Cite

View full text Add to dashboard Cite

Dengue virus (DENV) causes frequent epidemics infecting ϳ390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/ 36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (ϳ25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with Ͼ97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells. IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachiamediated suppression of DENV transmission.Citation Teramoto T, Huang X, Armbruster PA, Padmanabhan R. 2019. Infection of Aedes albopictus mosquito C6/36 cells with the wMelpop strain of Wolbachia modulates dengue virus-induce...

show abstract

Section: Cell Culture and Virus Infectionsmentioning

confidence: 99%

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

Teramoto

Huang

Armbruster

et al. 2019

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…At day 6 post-transfection, DENV4 POL chimeras did not show any detectable immunofluorescence-positive cells (Fig. 1B), similar to the exchange of DENV2 MTase with that of DENV4 domain (33). It required serial passages of DENV2 chimera RNA-transfected cells for ϳ30 days for DENV4 POL(264 -900aa) or longer for DENV4 POL(271-900aa) (ϳ42 days) to achieve replication fitness (Fig.…”

Section: Denv2 Ns5 Chimera Containing the Denv4 Ns5 Pol Domain Is Repmentioning

confidence: 65%

mentioning

confidence: 97%

“…In a previous study, we presented evidence that a functional interaction between the MTase and POL domains of DENV2 NS5 is required for viral replication (33). Even though DENV2 and DENV4 NS5 share 74% sequence identity, the replacement of NS5 MTase(1-270aa) with the corresponding region of DENV4 severely attenuates the viral replication (33). By repeated passages of chimera DENV2 RNA-transfected BHK-21 cells for ϳ30 days, a replication-competent chimera virus was evolved by an adaptive mutation, K74I, in the DENV4 NS5 MTase domain or D290N in the DENV2 NS3 helicase domain.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication

Teramoto

Balasubramanian

Choi

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer.

show abstract

“…Each Motif V mutation was designed to interrupt specific WT residue-residue interactions or residue-substrate interactions. As a negative control, D664 in the NS5 polymerase catalytic active site was mutated to valine (D664V) in order to disrupt viral genome replication (42)(43)(44)(45). Additionally, the NS3 mutants A286L (Motif II) and R387M (Motif IVa) were used as controls for ablating ATPase activity and RNA binding affinity, respectively (30,(46)(47)(48)(49)(50).…”

Section: Motif V Mutants Affect Viral Genome Replication -As Previousmentioning

confidence: 99%

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

Davidson

McCullagh

et al. 2019

Preprint

View full text Add to dashboard Cite

The unwinding of double-stranded RNA intermediates is a critical component for the replication of flavivirus RNA genomes. This function is achieved by the C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP and RNA binding pockets is not well understood. Previous molecular dynamics simulations published by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. In order to investigate the role of Motif V in this process, a study combining molecular dynamics, biochemistry and virology has been employed.Mutations of Motif V were tested in both replicon and recombinant protein systems to investigate viral genome replication, RNA binding affinity, ATP hydrolysis activity and helicase unwinding activity. Using these analyses, we found that T407A and S411A in Motif V demonstrated increased turnover rates, suggesting that the mutations causes the helicase to unwind dsRNA more quickly than WT. Additionally, simulations of each mutant were used to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP binding pocket and the helical gate. These data help define the linkage between ATP hydrolysis and helicase activity within NS3 and provide insight into the biophysical mechanisms for ATPase driven NS3 helicase function. Arthropod-borne flaviviruses, such as yellow fever virus, Japanese encephalitis virus, Zika virus, WestNile virus (WNV) and dengue virus, are a major health concern in the tropical and subtropical regions of the world (1, 2). Infection from these viruses cause disease symptoms ranging from flu-like illness to encephalitis, hemorrhagic fever, coma, and potentially death (3). Over half of the world population is at risk for infection from one or more of these viruses (4, 5). Dengue virus, specifically, infects around 50 million people each year, and of those individuals, 20,000 contract dengue hemorrhagic fever leading to their mortality (6). Additionally, WNV over the past 20 years within the 48 continental United States has around 50,000 clinical infections with a 5% mortality rate (7-9). Currently, there are no approved antiviral drugs for treating flaviviral infections and the vaccines in circulation, like the yellow fever vaccine, are not readily available worldwide for most flaviviruses (10-12). In order to develop new antiviral treatments (drugs and vaccines) for these viruses, a fundamental understanding of how these flaviviruses replicate is required.

show abstract

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

Cited by 20 publications

References 52 publications

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

Contact Info

Product

Resources

About