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Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Nayak A, Berry B, Tassetto M, Kunitomi M, Acevedo A, Deng C, Krutchinsky A, Gross J, Antoniewski C, Andino R - Nat. Struct. Mol. Biol. (2010)

Bottom Line: In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing.In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A.We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California, San Francisco, California, USA.

ABSTRACT
Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

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RNAi suppressors determine spread and pathogenesis of Sindbis virus in fruit fly. (a) Expression of the Sindbis virus structural proteins is regulated by a sub-genomic promoter (Pr1). CrPV-1A and DCV-1A were cloned under the control of a duplicated subgenomic promoter (Pr2) in the 3’ untranslated region (UTR) of the sindbis genome. These recombinant viruses also express green fluorescence protein (GFP) that cleaves at both N and C- terminus during structural polyprotein processing15. (b) BHK cells were infected with recombinant Sindbis viruses at MOI 1, virus sample were collected over a period of 24 hrs (h). Titer of the each sample [plaque forming unit (PFU) per ml] was measured by plaque assay and replication kinetics of each virus was determined. (c) Intrathoracic injections of flies with engineered Sindbis virus expressing CrPV-1A (Sin-CrPV148) induced higher mortality and tissue spread at similar dose of infection (2500–5000 PFU) compared to Sindbis virus expressing DCV-1A (Sin-DCV99). Survival rate of flies (%) was monitored daily (d) for thirty days. (d) (i) Flies injected with recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99 and Sin-DCVK73A) were imaged by GFP microscopy. For western blot analysis 4–5 flies from each injection at 11 days post infection were homogenized and blotted with anti-GFP antibodies (α-GFP). The same blot was stripped and probed with anti-actin antibody (α-actin). (ii) GFP fluorescence (AU) for recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99) was measured and data represents the means and standard error of the mean for 4–5 individual flies.
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Figure 6: RNAi suppressors determine spread and pathogenesis of Sindbis virus in fruit fly. (a) Expression of the Sindbis virus structural proteins is regulated by a sub-genomic promoter (Pr1). CrPV-1A and DCV-1A were cloned under the control of a duplicated subgenomic promoter (Pr2) in the 3’ untranslated region (UTR) of the sindbis genome. These recombinant viruses also express green fluorescence protein (GFP) that cleaves at both N and C- terminus during structural polyprotein processing15. (b) BHK cells were infected with recombinant Sindbis viruses at MOI 1, virus sample were collected over a period of 24 hrs (h). Titer of the each sample [plaque forming unit (PFU) per ml] was measured by plaque assay and replication kinetics of each virus was determined. (c) Intrathoracic injections of flies with engineered Sindbis virus expressing CrPV-1A (Sin-CrPV148) induced higher mortality and tissue spread at similar dose of infection (2500–5000 PFU) compared to Sindbis virus expressing DCV-1A (Sin-DCV99). Survival rate of flies (%) was monitored daily (d) for thirty days. (d) (i) Flies injected with recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99 and Sin-DCVK73A) were imaged by GFP microscopy. For western blot analysis 4–5 flies from each injection at 11 days post infection were homogenized and blotted with anti-GFP antibodies (α-GFP). The same blot was stripped and probed with anti-actin antibody (α-actin). (ii) GFP fluorescence (AU) for recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99) was measured and data represents the means and standard error of the mean for 4–5 individual flies.

Mentions: Virus pathogenesis is determined by many factors, including the ability of the virus to evade host immune response. Given the different properties of each suppressor, we considered whether they might determine the outcome of infection. Ideally, this hypothesis should be tested by engineering a virus carrying either of the different RNAi suppressors in an otherwise isogenic backgrounds. Given that no infectious clones are available for either DCV or CrPV, we examined the effect of CrPV-1A or DCV-1A in the context of a recombinant Sindbis virus infection. We engineered recombinant Sindbis viruses to express a green fluorescent protein (GFP) and RNAi suppressors CrPV-1A (Sin-CrPV148) or DCV-1A (Sin-DCV99) under the control of duplicated sub-genomic promoter (Fig. 6a)15. Control Sindbis viruses expressed inactive suppressors -- a truncated version of CrPV-1A (Sin-CrPV108) or DCV-1A carrying a mutation in the DSRM (Sin-DCVK73A)22. In addition to infecting insect cells, Sindbis also infects mammalian cells, which do not appear to use RNAi to control the virus, affording an unbiased control system for viral replication efficiency. No appreciable differences in virus replication kinetics were observed for these recombinant viruses in mammalian BHK-21 cells (Fig. 6b), indicating that expression of RNAi suppressors does not affect virus fitness in cells where the RNAi machinery does not play a major role in antiviral defense38.


Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Nayak A, Berry B, Tassetto M, Kunitomi M, Acevedo A, Deng C, Krutchinsky A, Gross J, Antoniewski C, Andino R - Nat. Struct. Mol. Biol. (2010)

RNAi suppressors determine spread and pathogenesis of Sindbis virus in fruit fly. (a) Expression of the Sindbis virus structural proteins is regulated by a sub-genomic promoter (Pr1). CrPV-1A and DCV-1A were cloned under the control of a duplicated subgenomic promoter (Pr2) in the 3’ untranslated region (UTR) of the sindbis genome. These recombinant viruses also express green fluorescence protein (GFP) that cleaves at both N and C- terminus during structural polyprotein processing15. (b) BHK cells were infected with recombinant Sindbis viruses at MOI 1, virus sample were collected over a period of 24 hrs (h). Titer of the each sample [plaque forming unit (PFU) per ml] was measured by plaque assay and replication kinetics of each virus was determined. (c) Intrathoracic injections of flies with engineered Sindbis virus expressing CrPV-1A (Sin-CrPV148) induced higher mortality and tissue spread at similar dose of infection (2500–5000 PFU) compared to Sindbis virus expressing DCV-1A (Sin-DCV99). Survival rate of flies (%) was monitored daily (d) for thirty days. (d) (i) Flies injected with recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99 and Sin-DCVK73A) were imaged by GFP microscopy. For western blot analysis 4–5 flies from each injection at 11 days post infection were homogenized and blotted with anti-GFP antibodies (α-GFP). The same blot was stripped and probed with anti-actin antibody (α-actin). (ii) GFP fluorescence (AU) for recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99) was measured and data represents the means and standard error of the mean for 4–5 individual flies.
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Figure 6: RNAi suppressors determine spread and pathogenesis of Sindbis virus in fruit fly. (a) Expression of the Sindbis virus structural proteins is regulated by a sub-genomic promoter (Pr1). CrPV-1A and DCV-1A were cloned under the control of a duplicated subgenomic promoter (Pr2) in the 3’ untranslated region (UTR) of the sindbis genome. These recombinant viruses also express green fluorescence protein (GFP) that cleaves at both N and C- terminus during structural polyprotein processing15. (b) BHK cells were infected with recombinant Sindbis viruses at MOI 1, virus sample were collected over a period of 24 hrs (h). Titer of the each sample [plaque forming unit (PFU) per ml] was measured by plaque assay and replication kinetics of each virus was determined. (c) Intrathoracic injections of flies with engineered Sindbis virus expressing CrPV-1A (Sin-CrPV148) induced higher mortality and tissue spread at similar dose of infection (2500–5000 PFU) compared to Sindbis virus expressing DCV-1A (Sin-DCV99). Survival rate of flies (%) was monitored daily (d) for thirty days. (d) (i) Flies injected with recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99 and Sin-DCVK73A) were imaged by GFP microscopy. For western blot analysis 4–5 flies from each injection at 11 days post infection were homogenized and blotted with anti-GFP antibodies (α-GFP). The same blot was stripped and probed with anti-actin antibody (α-actin). (ii) GFP fluorescence (AU) for recombinant Sindbis viruses (Sin-CrPV148, Sin-CrPV108, Sin-DCV99) was measured and data represents the means and standard error of the mean for 4–5 individual flies.
Mentions: Virus pathogenesis is determined by many factors, including the ability of the virus to evade host immune response. Given the different properties of each suppressor, we considered whether they might determine the outcome of infection. Ideally, this hypothesis should be tested by engineering a virus carrying either of the different RNAi suppressors in an otherwise isogenic backgrounds. Given that no infectious clones are available for either DCV or CrPV, we examined the effect of CrPV-1A or DCV-1A in the context of a recombinant Sindbis virus infection. We engineered recombinant Sindbis viruses to express a green fluorescent protein (GFP) and RNAi suppressors CrPV-1A (Sin-CrPV148) or DCV-1A (Sin-DCV99) under the control of duplicated sub-genomic promoter (Fig. 6a)15. Control Sindbis viruses expressed inactive suppressors -- a truncated version of CrPV-1A (Sin-CrPV108) or DCV-1A carrying a mutation in the DSRM (Sin-DCVK73A)22. In addition to infecting insect cells, Sindbis also infects mammalian cells, which do not appear to use RNAi to control the virus, affording an unbiased control system for viral replication efficiency. No appreciable differences in virus replication kinetics were observed for these recombinant viruses in mammalian BHK-21 cells (Fig. 6b), indicating that expression of RNAi suppressors does not affect virus fitness in cells where the RNAi machinery does not play a major role in antiviral defense38.

Bottom Line: In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing.In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A.We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of California, San Francisco, California, USA.

ABSTRACT
Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

Show MeSH
Related in: MedlinePlus