Involvement of the Host Cell Nuclear Envelope Membranes in the Replication of Japanese Encephalitis Virus

Zebovitz, Eugene; Leong, Jenny; Doughty, Sarah

doi:10.1128/iai.10.1.204-211.1974

Cited by 28 publications

(21 citation statements)

References 24 publications

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“…Interestingly, ϳ30 to 40% of the RdRp activity was associated with the nuclear fractions in all three flaviviruses studied ( Fig. 1A, lanes 1, 5, and 9), as reported earlier for JEV (45,54). Preparations of these nuclei were ascertained to be free of cytosolic contamination and more than 97% pure based on the absence of LDH (data not shown).…”

Section: Nuclear Association Of Viral Rc In Flavivirus-infected Cellssupporting

confidence: 81%

“…The RdRp activity of several flaviviruses including KUNV, DENV, JEV, and WNV has been established to be tightly associated with intracellular cytosolic membranes in numerous studies through biochemical (7,15,16,45,46,51,54) and ultrastructural (52) analyses. This has led most investigators to use postnuclear supernatant fractions as a source of mem-brane-bound flaviviral RC in in vitro RdRp assays.…”

mentioning

confidence: 99%

“…This has led most investigators to use postnuclear supernatant fractions as a source of mem-brane-bound flaviviral RC in in vitro RdRp assays. However, a few early studies employing in vivo labeling of JEV and WNV RNA have suggested that, in addition to cytoplasm, the viral RNA species is also found associated with the nuclear fraction of infected cells (16,45,54). Consequently, the outer nuclear envelope membrane (ONEM) and its extensions into the endoplasmic reticulum (ER) were also proposed to harbor the flavivirus RC.…”

mentioning

confidence: 99%

“…Flaviviruses have been demonstrated to involve host nuclei in their replication, albeit indirectly. Treatment of host cells with either actinomycin D or alpha-amanitin before virus infection compromised viral replication and subsequently viral titers (49,54). Enucleation of cells during the latent period of virus infection also inhibited JEV replication (29).…”

mentioning

confidence: 99%

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Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

Uchil

Kumar

Satchidanandam

2006

J Virol

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Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membranebound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.Several members of the genus Flavivirus, which comprises viruses with a single-strand RNA of positive polarity, such as West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), Murray Valley encephalitis virus and tick-borne encephalitis virus, are pathogens of humans and animals. The 11-kb-long flaviviral genomes encode three structural and seven nonstructural proteins, derived by processing of the primary polyprotein translation product by host signalases and a viral protease, NS3. The NS3 protein, in addition, has helicase and NTPase functions in flaviviral replication. The viral RNA-dependent RNA polymerase (RdRp), the product of the viral NS5 gene, is responsible for replication of the viral genome within putative complexes comprising both viral and an as-yet-unidentified host protein(s). The replication complex (RC) uses the genomic RNA to generate a double-stranded replicative form (RF), and initiation of RNA synthesis on this template results in formation of replicative intermediates (RI) that resolve, upon completion of strand synthesis, to produce a single-stranded viral RNA (vRNA) and RF. This model of flavivirus replication by an asymmetric and semiconservative mode was first proposed for Kunjin virus (KUNV) (8) and has been subsequently confirmed for DENV (9) and JEV (49).The RdRp activity of several flaviviruses including KUNV, DENV, JEV, and WNV has been established to be tightly associated with intracellular cytosolic membranes in numerous studies through biochemical (7,15,16,45,46,51,54) and ultrastructural (52) analyses. This has led ...

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Section: Nuclear Association Of Viral Rc In Flavivirus-infected Cellssupporting

confidence: 81%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

Uchil

Kumar

Satchidanandam

2006

J Virol

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“…Some negative-stranded RNA viruses use the host cytoplasm as the primary site of replication, they can also recruit nuclear factors and alter their nuclear-cytoplasm trafficking to enhance viral replication (Hiscox, 2003). The host nucleus proteins could be recruited to cytoplasm to facilitate flaviviruses replication (Uchil and Satchidanandam, 2003b;Zebovitz et al, 1974). It has been reported that DDX5 is redistributed into the cytoplasm during HCV infection (Goh et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

et al. 2013

Antiviral Research

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Japanese encephalitis virus (JEV), one of the causes for epidemic encephalitis, belongs to the family of Flaviviridae. In this study, we demonstrated that cellular DEAD-box RNA helicase DDX5 plays an important role in JEV replication. The knockdown of DDX5 was able to decrease JEV replication, and overexpression of DDX5 mutants lacking the helicase activity also reduced JEV replication, suggesting the helicase activity is essential for JEV replication. DDX5 knockdown did not affect virus assembly and release. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX5 could interact with JEV core protein, non-structural protein 3 (NS3) and 5 (NS5-MTase and NS5-RdRp domains). Meanwhile, we also confirmed that DDX5 interacts with these viral proteins during JEV infection. Confocal microscopy analysis showed that endogenous DDX5 is recruited to the cytoplasm and colocalizes with these viral proteins and viral RNA. RNA-pulldown experiment showed that DDX5 only binds to the JEV 3' untranslated region (UTR). Finally, we confirmed the role of DDX5 in JEV RNA replication using JEV-replicon system. In conclusion, we identified DDX5 as a positive regulator for JEV replication.

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Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

et al. 2019

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For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed.

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Involvement of the Host Cell Nuclear Envelope Membranes in the Replication of Japanese Encephalitis Virus

Cited by 28 publications

References 24 publications

Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

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