Viral RNA Accumulation Research Articles

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.

Modulation of Hepatitis C Virus RNA Accumulation and Translation by DDX6 and miR-122 Are Mediated by Separate Mechanisms

DDX6 and other P-body proteins are required for efficient replication of Hepatitis C Virus (HCV) by unknown mechanisms. DDX6 has been implicated in miRNA induced gene silencing, and since efficient HCV replication and translation relies on the cellular microRNA, miR-122, we hypothesized that DDX6 had a role in the mechanism of action of miR-122. However, by using multiple HCV translation and replication assays we have found this is not the case. DDX6 silencing decreased HCV replication and translation, but did not affect the ability of miR-122 to stimulate HCV translation or promote HCV RNA accumulation. In addition, the negative effect of DDX6 silencing on HCV replication and translation was not dependent on miR-122 association with the HCV genome. Thus, DDX6 does not have a role in the activity of miR-122, and it appears that DDX6 and miR-122 modulate HCV through distinct pathways. This effect was seen in both Huh7.5 cells and in Hep3B cells, indicating that the effects are not cell type specific. Since infections by other viruses in the Flaviviridae family, including Dengue and West Nile Virus, also disrupt P-bodies and are regulated by DDX6, we speculate that DDX6 may have a common function that support the replication of several Flaviviruses.

Maintaining the structural integrity of the bamboo mosaic virus3′ untranslated region is necessary for retaining the catalytic constant for minus-strand RNA synthesis

BackgroundBamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus and have a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genome was mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMV replicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA to initiate minus-strand RNA synthesis in vitro.ResultsTo investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo, we constructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used to replace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX 3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotiana benthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generated by the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, the Vmax/KM of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strand RNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequately replicated in vitro.ConclusionsAmong the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that contained non-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation in protoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA was not interrupted and the domains were not replaced with the 3′ UTR of PVX RNA in vivo.

The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection.In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation.The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3′ untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis.The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis.

Modulation of GB Virus B RNA Abundance by MicroRNA-122: Dependence on and Escape from MicroRNA-122 Restriction

Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5'-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.

Inhibition of Cellular Autophagy Deranges Dengue Virion Maturation

Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

Cucumber vein yellowing virus isolate-specific expression of symptoms and viral RNA accumulation in susceptible and resistant cucumber cultivars

Since its outbreak in Spain during the fall of 2000, Cucumber vein yellowing virus (CVYV) has become into a limiting factor in cucurbit cultivation in this country, particularly in cucumber. The impact of the virus has been limited after the introduction of resistant cucumber cultivars and the implementation of cultural practices to control whitefly, the virus vector. In Spain, CVYV isolates are genetically similar and cucumber resistance breaking isolates have not been reported. A CVYV isolate from Jordan (Jor) was previously reported as being more virulent in cucumber cv. Beit Alfa than an isolate from Israel. To study the reactions of different cucumber cultivars to the virulent Jor isolate in comparison with a Spanish isolate of (Alm 1A), we have inoculated both isolates in 10 commercial cucumber cultivars with resistance to the virus. Also, we have used real-time RT-PCR to estimate virus accumulation of Jor and Alm 1A isolates in three of these cultivars, one described as susceptible and two as resistant to CVYV. The Spanish Alm 1A isolate caused mild or no symptoms in resistant cultivars whereas Jor isolate caused intense symptoms in all cultivars. Alm1 A genomic RNA accumulated similarly in the three cultivars, whereas the accumulation of Jor genomic RNA was lower in the resistant cultivars than in the susceptible cultivar. Also the Jor isolate and accumulated earlier and at a higher level than the Alm 1A isolate in the three cultivars.

Variations in Hypovirus Interactions with the Fungal-Host RNA-Silencing Antiviral-Defense Response

Hypoviruses Cryphonectria hypovirus 1 (CHV-1)/EP713, CHV-1/Euro7, and CHV-1/EP721, which infect the chestnut blight fungus Cryphonectria parasitica, differ in their degrees of virulence attenuation (hypovirulence), symptom expression, and viral RNA accumulation, even though they share between 90% and 99% amino acid sequence identity. In this report we examine whether this variability is influenced by interactions with the C. parasitica Dicer gene dcl2-dependent RNA-silencing antiviral defense response. The mild symptoms exhibited by strains infected with CHV-1/Euro7 and CHV-1/EP721 relative to those with severe hypovirus CHV-1/EP713 did not correlate with a higher induction of the RNA-silencing pathway. Rather, dcl2 transcripts accumulated to a higher level (∼8-fold) following infection by CHV-1/EP713 than following infection by CHV-1/Euro7 (1.2-fold) or CHV-1/EP721 (1.4-fold). The differences in dcl2 transcript accumulation in response to CHV-1/EP713 and CHV-1/EP721 were unrelated to the suppressor of RNA silencing, p29, encoded by the two viruses. Moreover, the coding strand viral RNA levels increased by 33-, 32-, and 16-fold for CHV-1/EP713, CHV-1/Euro7, and CHV-1/EP721, respectively, in Δdcl2 mutant strains. This indicates that a very robust antiviral RNA-silencing response was induced against all three viruses, even though significant differences in the levels of dcl2 transcript accumulation were observed. Unexpectedly, the severe debilitation previously reported for CHV-1/EP713-infected Δdcl2 mutant strains, and observed here for the CHV-1/Euro7-infected Δdcl2 mutant strains, was not observed with infection by CHV-1/EP721. By constructing chimeric viruses containing portions of CHV-1/EP713 and CHV-1/EP721, it was possible to map the region that is associated with the severe debilitation of the Δdcl2 mutant hosts to a 4.1-kb coding domain located in the central part of the CHV-1/EP713 genome.

The E2-E166K substitution restores Chikungunya virus growth in OAS3 expressing cells by acting on viral entry

Human 2′,5′-oligoadenylate synthetase 3 (OAS3) exerts antiviral effect against alphaviruses including Chikungunya virus (CHIKV) by inhibiting viral RNA accumulation. Here, we identified a CHIKV variant exhibiting a remarkable resistance to the antiviral action of OAS3 in human epithelial HeLa cells. Using a molecular clone of CHIKV with Renilla luciferase inserted as a reporter gene in the non-structural region, we demonstrated that a single glutamine-to-lysine amino acid change at position 166 of the envelope E2 glycoprotein restores CHIKV replication in OAS3 expressing HeLa cells. Viral entry assays showed that CHIKV with a lysine at position E2-166 was more efficient at entering the replicative pathway. The E2-E166K substitution promotes a greater efficiency of CHIKV replication in human myoblasts leading to severe apoptosis through a more robust activation of the PKR pathway. These observations provide a new insight into the role of E2 into the pathogenicity of CHIKV in human cells.

The R-rich motif of Beet black scorch virus P7a movement protein is important for the nuclear localization, nucleolar targeting and viral infectivity

Beet black scorch virus (BBSV) encodes three movement proteins (P7a, P7b and P5′) that facilitate its cell-to-cell movement. An arginine-rich motif of P7a N-terminus was found to determine nuclear and nucleolar localization. Amino acids substitution or deletion of the R-rich motif interfered with P7a nuclear and nucleolar localization. Bimolecular fluorescence complementation (BiFC) assays revealed that P7a protein interacted with Nicotiana benthamiana nuclear import factor importin α, suggesting that P7a is translocated into the nucleus by the classical importin α/β-dependent pathway. Moreover, P7a also interacted with the nucleolar protein fibrillarin. Mutations in the R-rich motif of P7a diminished P7a interactions with importin α and fibrillarin, influenced viral replication in Nicotiana benthamiana protoplasts and altered the symptom phenotype and viral RNA accumulation in Chenopodium amaranticolor plants. These results demonstrate that the R-rich motif of P7a is correlated with nuclear and nucleolar localization, viral replication and virus infection.

A game-theoretic model of interactions between Hibiscus latent Singapore virus and tobacco mosaic virus.

Mixed virus infections in plants are common in nature and their interactions affecting host plants would depend mainly on plant species, virus strains, the order of infection and initial amount of inoculum. Hence, the prediction of outcome of virus competition in plants is not easy. In this study, we applied evolutionary game theory to model the interactions between Hibiscus latent Singapore virus (HLSV) and Tobacco mosaic virus (TMV) in Nicotiana benthamiana under co-infection in a plant host. The accumulation of viral RNA was quantified using qPCR at 1, 2 and 8 days post infection (dpi), and two different methods were employed to predict the dominating virus. TMV was predicted to dominate the game in the long run and this prediction was confirmed by both qRT-PCR at 8 dpi and the death of co-infected plants after 15 dpi. In addition, we validated our model by using data reported in the literature. Ten out of fourteen reported co-infection outcomes agreed with our predictions. Explanations were given for the four interactions that did not agree with our model. Hence, it serves as a valuable tool in making long term predictions using short term data obtained in virus co-infections.

Modeling the Intracellular Dynamics of Influenza Virus Replication To Understand the Control of Viral RNA Synthesis

Influenza viruses transcribe and replicate their negative-sense RNA genome inside the nucleus of host cells via three viral RNA species. In the course of an infection, these RNAs show distinct dynamics, suggesting that differential regulation takes place. To investigate this regulation in a systematic way, we developed a mathematical model of influenza virus infection at the level of a single mammalian cell. It accounts for key steps of the viral life cycle, from virus entry to progeny virion release, while focusing in particular on the molecular mechanisms that control viral transcription and replication. We therefore explicitly consider the nuclear export of viral genome copies (vRNPs) and a recent hypothesis proposing that replicative intermediates (cRNA) are stabilized by the viral polymerase complex and the nucleoprotein (NP). Together, both mechanisms allow the model to capture a variety of published data sets at an unprecedented level of detail. Our findings provide theoretical support for an early regulation of replication by cRNA stabilization. However, they also suggest that the matrix protein 1 (M1) controls viral RNA levels in the late phase of infection as part of its role during the nuclear export of viral genome copies. Moreover, simulations show an accumulation of viral proteins and RNA toward the end of infection, indicating that transport processes or budding limits virion release. Thus, our mathematical model provides an ideal platform for a systematic and quantitative evaluation of influenza virus replication and its complex regulation.

Virus-induced Silencing of the WRKY1 Transcription Factor that Interacts with the SL1 Structure of Potato virus X Leads to Higher Viral RNA Accumulation and Severe Necrotic Symptoms

<TEX>$Potato$</TEX> <TEX>$virus$</TEX> <TEX>$X$</TEX> (PVX) replication is precisely regulated by regulatory viral sequences and by viral and/or host proteins. In a previous study, we identified a 54-kDa cellular tobacco protein that bound to a region within the first 46 nucleotides (nt) of the 5' non-translated region (NTR) of the viral genome. Optimal binding was dependent upon the presence of an ACCA sequence at nt 10-13. To identify host factors that bind to 5' NTR elements including AC-rich sequences as well as stemloop 1 (SL1), we used northwestern blotting and matrixassisted laser desorption/ionization time-of-flight mass spectrometry for peptide mass fingerprinting. We screened several host factors that might affect PVX replication and selected a candidate protein, <TEX>$Nicotiana$</TEX> <TEX>$tabacum$</TEX> WRKY transcription factor 1 (NtWRKY1). We used a <TEX>$Tobacco$</TEX> <TEX>$rattle$</TEX> <TEX>$virus$</TEX> (TRV)-based virus-induced gene silencing (VIGS) system to investigate the role of NtWRKY1 in PVX replication. Silencing of <TEX>$WRKY1$</TEX> in <TEX>$Nicotiana$</TEX> <TEX>$benthamiana$</TEX> caused lethal apical necrosis and allowed an increase in PVX RNA accumulation. This result could reflect the balancing of PVX accumulation in a systemic <TEX>$N.$</TEX> <TEX>$benthamiana$</TEX> host to maintain PVX survival and still produce a suitable appearance of mosaic and mottle symptoms. Our results suggest that PVX may recruit the WRKY transcription factor, which binds to the 5' NTR of viral genomic RNA and acts as a key regulator of viral infection.

A single amino acid in the helicase domain of PMMoV-S is responsible for its enhanced accumulation in C. chinense (L3L3) plants at 32 °C

In Capsicum chinense (L3L3) plants a higher accumulation of the tobamovirus Pepper mild mottle virus strain S (PMMoV-S) as compared to the Italian strain PMMoV-I is detected when plants are grown at 32°C. By using a reverse genetic approach, we have established that a single amino acid at position 898 in the helicase domain of the polymerase protein, outside of the conserved regions of the helicase, is critical for the higher accumulation of PMMoV-S observed. It also is necessary for both increased accumulation of viral RNA of both polarities in pepper protoplasts and enhanced cell-to-cell movement in C. chinense plants. The influence of thermoresistance of PMMoV-S, a P1,2 pathotype, and its prevalence on pepper cultivars over PMMoV-I, a P1,2,3, pathotype, is discussed.

Microarray Analysis Shows That Recessive Resistance to Watermelon mosaic virus in Melon Is Associated with the Induction of Defense Response Genes

Resistance to Watermelon mosaic virus (WMV) in melon (Cucumis melo L.) accession TGR-1551 is characterized by a significant reduction in virus titer, and is inherited as a recessive, loss-of-susceptibility allele. We measured virus RNA accumulation in TGR-1551 plants and a susceptible control ('Tendral') by real-time quantitative polymerase chain reaction, and also profiled the expression of 17,443 unigenes represented on a melon microarray over a 15-day time course. The virus accumulated to higher levels in cotyledons of the resistant variety up to 9 days postinoculation (dpi) but, thereafter, levels increased in the susceptible variety while those in the resistant variety declined. Microarray experiments looking at the early response to infection (1 and 3 dpi), as well as responses after 7 and 15 dpi, revealed more profound transcriptomic changes in resistant plants than susceptible ones. The gene expression profiles revealed deep and extensive transcriptome remodeling in TGR-1551 plants, often involving genes with pathogen response functions. Overall, our data suggested that resistance to WMV in TGR-1551 melon plants is associated with a defense response, which contrasts with the recessive nature of the resistance trait.

Evolution of a Species-Specific Determinant within Human CRM1 that Regulates the Post-transcriptional Phases of HIV-1 Replication

The human immunodeficiency virus type-1 (HIV-1) Rev protein regulates the nuclear export of intron-containing viral RNAs by recruiting the CRM1 nuclear export receptor. Here, we employed a combination of functional and phylogenetic analyses to identify and characterize a species-specific determinant within human CRM1 (hCRM1) that largely overcomes established defects in murine cells to the post-transcriptional stages of the HIV-1 life cycle. hCRM1 expression in murine cells promotes the cytoplasmic accumulation of intron-containing viral RNAs, resulting in a substantial stimulation of the net production of infectious HIV-1 particles. These stimulatory effects require a novel surface-exposed element within HEAT repeats 9A and 10A, discrete from the binding cleft previously shown to engage Rev's leucine-rich nuclear export signal. Moreover, we show that this element is a unique feature of higher primate CRM1 proteins, and discuss how this sequence has evolved from a non-functional, ancestral sequence.

The Splicing Factor Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection.

Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2149 (VP2149M) or glutamine to glutamic acid substitution at VP1145 (VP1145E) showed greater viral titers and apoptosis. Synergistic effect of VP2149M and VP1145E double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2149M and VP1145E mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

The DEAD-box RNA Helicase DDX6 is Required for Efficient Encapsidation of a Retroviral Genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging.

Dynamics of alternative modes of RNA replication for positive-sense RNA viruses

We propose and study nonlinear mathematical models describing the intracellular time dynamics of viral RNA accumulation for positive-sense single-stranded RNA viruses. Our models consider different replication modes ranging between two extremes represented by the geometric replication (GR) and the linear stamping machine replication (SMR). We first analyse a model that quantitatively reproduced experimental data for the accumulation dynamics of both polarities of turnip mosaic potyvirus RNAs. We identify a non-degenerate transcritical bifurcation governing the extinction of both strands depending on three key parameters: the mode of replication (α), the replication rate (r) and the degradation rate (δ) of viral strands. Our results indicate that the bifurcation associated with α generically takes place when the replication mode is closer to the SMR, thus suggesting that GR may provide viral strands with an increased robustness against degradation. This transcritical bifurcation, which is responsible for the switching from an active to an absorbing regime, suggests a smooth (i.e. second-order), absorbing-state phase transition. Finally, we also analyse a simplified model that only incorporates asymmetry in replication tied to differential replication modes.