Inhibition of Vaccinia Virus Replication by Two Small Interfering RNAs Targeting B1R and G7L Genes and Their Synergistic Combination with Cidofovir

ABSTRACT In view of the threat of the potential use of variola virus in a terrorist attack, considerable efforts have been performed to develop new antiviral strategies against orthopoxviruses. Here we report on the use of RNA interference, either alone or in combination with cidofovir, as an approach to inhibit orthopoxvirus replication. Two selected small interfering RNAs (siRNAs), named siB1R-2 and siG7L-1, and a previously reported siRNA, i.e., siD5R-2 (which targets the viral D5R mRNA), were evaluated for antiviral activity against vaccinia virus (VACV) by plaque reduction and virus yield assays. siB1R-2 and siG7L-1, administered before or after viral infection, reduced VACV replication by more than 90%. Also, these two siRNAs decreased monkeypox virus replication by 95% at a concentration of 1 nM. siB1R-2 and siG7L-1 were demonstrated to specifically silence their corresponding transcripts, i.e., B1R and G7L mRNAs, without induction of a beta interferon response. Strong synergistic effects were observed when siB1R-2, siG7L-1, or siD5R-2 was combined with cidofovir. In addition, the antiviral activities of these three siRNAs were evaluated against VACV resistant to cidofovir and other acyclic nucleoside phosphonates. siG7L-1 and siD5R-2 remained active against four of five VACV mutants, while siB1R-2 showed activity against only one of the mutants. Our results showed that siRNAs are potent inhibitory agents in vitro, not only against wild-type VACV but also against several cidofovir-resistant VACV. Furthermore, we showed that a combined therapy using siRNA and cidofovir may be useful in the treatment of poxvirus infections.

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Inhibitors of the Interferon Response Enhance Virus Replication In Vitro

PLoS ONE ◽

10.1371/journal.pone.0112014 ◽

2014 ◽

Vol 9 (11) ◽

pp. e112014 ◽

Cited By ~ 33

Author(s):

Claire E. Stewart ◽

Richard E. Randall ◽

Catherine S. Adamson

Keyword(s):

Virus Replication ◽

Interferon Response

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The Nucleoside Triphosphatase and Helicase Activities of Vaccinia Virus NPH-II Are Essential for Virus Replication

Journal of Virology ◽

10.1128/jvi.72.6.4729-4736.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4729-4736 ◽

Cited By ~ 30

Author(s):

Christian H. Gross ◽

Stewart Shuman

Keyword(s):

Vaccinia Virus ◽

Virus Replication ◽

Rna Binding ◽

Rna Helicase ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Sequence Motifs ◽

Rna Unwinding

ABSTRACT Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-tsviruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.

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The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence

Journal of General Virology ◽

10.1099/0022-1317-83-4-873 ◽

2002 ◽

Vol 83 (4) ◽

pp. 873-878 ◽

Cited By ~ 9

Author(s):

Nicola Price ◽

David C. Tscharke ◽

Geoffrey L. Smith

Keyword(s):

Cell Culture ◽

Vaccinia Virus ◽

Virus Replication ◽

Lipid Membrane ◽

Signal Sequence ◽

Intracellular Protein ◽

Microsomal Membranes ◽

Infectious Cycle

Vaccinia virus (VV) strain Western Reserve gene B9R is shown to encode an intracellular 6 kDa protein that is expressed late during the infectious cycle. In vitro transcription and translation produced two polypeptides in the presence of microsomal membranes, but only the larger protein in the absence of membranes. The smaller protein sedimented with microsomes during centrifugation, suggesting it was inserted into the lipid membrane or into the microsomal lumen via the N-terminal hydrophobic signal sequence that was subsequently cleaved proteolytically. A VV mutant lacking B9R was constructed and found to replicate normally in cell culture and two in vivo models.

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Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

PLoS Pathogens ◽

10.1371/journal.ppat.1009790 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009790

Author(s):

Yuqiang Zhang ◽

Yan Xu ◽

Yunpeng Dai ◽

Zhe Li ◽

Jiaxing Wang ◽

...

Keyword(s):

Virus Infection ◽

Sindbis Virus ◽

De Novo ◽

Somatic Cells ◽

Interferon Response ◽

Small Interfering Rnas ◽

Rnai Suppressor ◽

Antiviral Function

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.

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Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.2.404-412.2004 ◽

2004 ◽

Vol 48 (2) ◽

pp. 404-412 ◽

Cited By ~ 111

Author(s):

Debra C. Quenelle ◽

Deborah J. Collins ◽

W. Brad Wan ◽

James R. Beadle ◽

Karl Y. Hostetler ◽

...

Keyword(s):

Single Dose ◽

Vaccinia Virus ◽

Virus Replication ◽

Oral Treatment ◽

Ether Lipid ◽

Cowpox Virus ◽

Virus Infections

ABSTRACT Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log10. In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.

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RNA interference and the use of small interfering RNA to study gene function in mammalian systems

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01582 ◽

2004 ◽

Vol 33 (3) ◽

pp. 545-557 ◽

Cited By ~ 92

Author(s):

I Bantounas ◽

L A Phylactou ◽

J B Uney

Keyword(s):

Rna Interference ◽

Gene Function ◽

Viral Vector ◽

Sirna Delivery ◽

Interferon Response ◽

Rnai Pathway ◽

Small Interfering Rnas ◽

Rnai Technology

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

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