IFN-deficient Cells Research Articles

A dsRNA-binding mutant reveals only a minor role of exonuclease activity in interferon antagonism by the arenavirus nucleoprotein.

The arenavirus nucleoprotein (NP) plays an important role in the virus' ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-β promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-β mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.

SARS-CoV-2 RNAs are processed into 22-nt vsRNAs in Vero cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic, resulting in great fatalities around the world. Although the antiviral roles of RNA interference (RNAi) have been well studied in plants, nematodes and insects, the antiviral roles of RNAi in mammalians are still debating as RNAi effect is suspected to be suppressed by interferon (IFN) signaling pathways in most cell types. To determine the role of RNAi in mammalian resistance to SARS-CoV-2, we studied the profiling of host small RNAs and SARS-CoV-2 virus-derived small RNAs (vsRNAs) in the early infection stages of Vero cells, an IFN-deficient cell line. We found that host microRNAs (miRNAs) were dysregulated upon SARS-CoV-2 infection, resulting in downregulation of microRNAs playing antiviral functions and upregulation of microRNAs facilitating viral proliferations. Moreover, vsRNA peaked at 22 nt at negative strand but not the positive strand of SARS-CoV-2 and formed successive Dicer-spliced pattern at both strands. Similar characteristics of vsRNAs were observed in IFN-deficient cell lines infected with Sindbis and Zika viruses. Together, these findings indicate that host cell may deploy RNAi pathway to combat SARS-CoV-2 infection in IFN-deficient cells, informing the alternative antiviral strategies to be developed for patients or tissues with IFN deficiency.

Decreased Virulence of Ross River Virus Harboring a Mutation in the First Cleavage Site of Nonstructural Polyprotein Is Caused by a Novel Mechanism Leading to Increased Production of Interferon-Inducing RNAs.

ABSTRACTInfection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo. Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease.

Abrogation of the interferon response promotes more efficient human cytomegalovirus replication.

The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to block either the production of or the response to type I IFNs. In IFN-deficient cells, HCMV produced larger plaques and spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.

The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

Identification of Amino Acid Residues Critical for the Anti-Interferon Activity of the Nucleoprotein of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis virus (LCVM) nucleoprotein (NP) counteracts the host type I interferon (IFN) response by inhibiting activation of the IFN regulatory factor 3 (IRF3). In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF

BackgroundThe competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells.ResultsIn IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66–67 aa).ConclusionOur data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

Cytokine-Independent Upregulation of MDA5 in Viral Infection

The RNA helicases RIG-I and MDA5 detect virus infection of dendritic cells (DCs) leading to cytokine induction. Maximal sensitivity for virus detection by these helicases is obtained after their upregulation, which is thought to occur primarily through type I interferon (IFN) signaling. Here we demonstrate that in response to paramyxovirus infection, RIG-I upregulation requires type I IFN whereas MDA5 expression is increased by Sendai virus infection independently of signaling mediated by type I IFN, STAT1, tumor necrosis factor alpha, or NF-kappaB. This MDA5 upregulation is largely lost in IRF3 knockout DCs and is achieved in type I IFN-deficient cells expressing constitutively active IRF3.