Overproduction of Double-Stranded RNA in Vesicular Stomatitis Virus-Infected Cells Activates a Constitutive Cell-Type-Specific Antiviral Response

104

As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-␤) very poorly, two unnatural SeV infections were used to study virus-induced IFN-␤ activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(؉/؊), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form doublestranded RNA (dsRNA) with capped 5 ends. We found that (i) virus-induced signaling to IFN-␤ depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-␤. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or ppp RNAs on IFN-␤ activation but also synergistically enhances these effects. SeV-V minus infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or ppp RNAs.All viruses evade the cellular innate immune system in part by expressing gene products that interfere with the ability of the host cell to establish an antiviral state (6). In the case of the Paramyxovirinae, this anti-host-defense activity is due mostly to viral C and V proteins (15,27,31). The C and V proteins are encoded by separate alternate open reading frames (ORFs), which both overlap that of the P protein. V and C are also referred to as accessory gene products, as not all members of this virus subfamily express one or the other. More specifically, rubulavirus and avulavirus express V but do not express C proteins, and human parainfluenza virus type 1 (PIV1), a respirovirus most closely related to Sendai virus (SeV), expresses C but does not express a V protein (16,20).Paramyxovirus V and C proteins antagonize interferon (IFN) signaling by various mechanisms, and they also target the production of type I IFN (15, 31). Beta IFN (IFN-␤) production is one of the earliest events in the cellular innate immune response, which leads to the establishment of an antiviral state. IFN-␤ production requires the coordinated activation of several transcription factors, including NF-B and IRF3 (15, 29). For intracellular RNA virus replication, the signaling pathway that leads to IRF3 activation starts with mda-5 and RIG-I, two cytoplasmic DExH/D-box helicases with N-terminal CARD domains. These helicases respond to doublestranded RNA (dsRNA) and, at least for RIG-I, to 5Ј-triphosphorylated single-stranded RNA (ssRNA) ( ppp RNA), which are generated in the cytoplasm during RNA virus replication (9,11,25). Upon the detection of these viral RNAs, the CARD domains of these helicases interact with IPS-1/C...

Section: Resultsmentioning

confidence: 99%

Activation of the Beta Interferon Promoter by Unnatural Sendai Virus Infection Requires RIG-I and Is Inhibited by Viral C Proteins

Strahle

Marq

Brini

et al. 2007

104

“…Pictures were taken with a Leica TCS-SP5 confocal microscope. 24). Hence, it would be interesting to test whether the observed growth restriction phenotype of this VSV polR mutant could be linked to the activity of UAP56.…”

Section: Discussionmentioning

confidence: 99%

The Cellular RNA Helicase UAP56 Is Required for Prevention of Double-Stranded RNA Formation during Influenza A Virus Infection

Wisskirchen

Ludersdorfer

Müller

et al. 2011

The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/ replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

“…It is therefore likely that the U1323A mutation impairs viral fitness by producing abnormal capsids with limited ability to support transcription and/or replication. The N protein forms a tunnel-like structure that wraps the viral genomic RNA and remains assembled during the entire infection cycle, shielding the viral RNA (16,20,33). Recent work showed, though, that conformational changes in the N protein make the RNA transiently accessible to other proteins to allow transcription and replication (19).…”

Section: Discussionmentioning

confidence: 99%

Relationship between within-Host Fitness and Virulence in the Vesicular Stomatitis Virus: Correlation with Partial Decoupling

Furió

Garijo

Durán

et al. 2012

e Given the parasitic nature of viruses, it is sometimes assumed that rates of viral replication and dissemination within hosts (within-host fitness) correlate with virulence. However, there is currently little empirical evidence supporting this principle. To test this, we quantified the fitness and virulence of 21 single-or double-nucleotide mutants of the vesicular stomatitis virus in baby hamster kidney cells (BHK-21). We found that, overall, these two traits correlated positively, but significant outliers were identified. Particularly, a single mutation in the conserved C terminus of the N nucleocapsid (U1323A) had a strongly deleterious fitness effect but did not alter or even slightly increased virulence. We also found a double mutant of the M matrix protein and G glycoprotein (U2617G/A3802G mutant) with high fitness yet low virulence. We further characterized these mutants in primary cultures from mouse brain cells and in vivo and found that their relative fitness values were similar to those observed in BHK-21 cells. The mutations had weak effects on the virus-induced death rate of total brain cells, although they specifically reduced neuron death rates. Furthermore, increased apoptosis levels were detected in neurons infected with the U2617G/A3802G mutant, consistent with its known inability to block interferon secretion. In vivo, this mutant had reduced virulence and, despite its low brain titer, it retained a relatively high fitness value owing to its ability to suppress competitor viruses. Overall, our results are in broad agreement with the notion that viral fitness and virulence should be positively correlated but show that certain mutations can break this association and that the fitness-virulence relationship can depend on complex virus-host and virus-virus interactions.