Actomyosin-driven motility and coalescence of phase-separated viral inclusion bodies are required for efficient replication of a plant rhabdovirus.

Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Function of Host Protein Staufen1 in Rabies Virus Replication.

Rabies virus is a highly neurophilic negative-strand RNA virus with high lethality and remains a huge public health problem in developing countries to date. The double-stranded RNA-binding protein Staufen1 (STAU1) has multiple functions in RNA virus replication, transcription, and translation. However, its function in RABV infection and its mechanism of action are not clear. In this study, we investigated the role of host factor STAU1 in RABV infection of SH-SY-5Y cells. Immunofluorescence, TCID50 titers, confocal microscopy, quantitative real-time PCR and Western blotting were carried out to determine the molecular function and subcellular distribution of STAU1 in these cell lines. Expression of STAU1 in SH-SY-5Y cells was down-regulated by RNA interference or up-regulated by transfection of eukaryotic expression vectors. The results showed that N proficiently colocalized with STAU1 in SH-SY-5Y at 36 h post-infection, and the expression level of STAU1 was also proportional to the time of infection. Down-regulation of STAU1 expression increased the number of Negri body-like structures, enhanced viral replication, and a caused 10-fold increase in viral titers. Meanwhile, N protein and G protein mRNA levels also accumulated gradually with increasing infection time, which implied that STAU1 inhibited rabies virus infection of SH-SY-5Y cells in vitro. In conclusion, our results provide important clues for the detailed replication mechanism of rabies virus and the discovery of therapeutic targets.

Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation.

Liquid-liquid phase separation (LLPS) can drive formation of diverse and essential macromolecular structures, including those specified by viruses. Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genomes associate with the viral encoded Latency-Associated Nuclear Antigen (LANA) to form stable nuclear bodies (NBs) during latent infection. Here, we show that LANA-NB formation and KSHV genome conformation involves LLPS. Using LLPS disrupting solvents, we show that LANA-NBs are partially disrupted, while DAXX and PML foci are highly resistant. LLPS disruption altered the LANA-dependent KSHV chromosome conformation but did not stimulate lytic reactivation. We found that LANA-NBs undergo major morphological transformation during KSHV lytic reactivation to form LANA-associated replication compartments encompassing KSHV DNA. DAXX colocalizes with the LANA-NBs during latency but is evicted from the LANA-associated lytic replication compartments. These findings indicate the LANA-NBs are dynamic super-molecular nuclear structures that partly depend on LLPS and undergo morphological transitions corresponding to the different modes of viral replication.

Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling.

Viruses routinely employ strategies to prevent the activation of innate immune signaling in infected cells. Respiratory syncytial virus (RSV) is no exception, as it encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signaling. However, RSV-encoded mechanisms for inhibiting NF-κB signaling are less well characterized. In this study, we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV-infected cells, we demonstrated that the p65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured p65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following tumor necrosis factor alpha (TNF-α) stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalize the RSV N protein and p65 within bovine RSV (bRSV) IBs, which are granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterization of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate-a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally, they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells.IMPORTANCE Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.

African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies.

African horse sickness virus (AHSV) is the causative agent of the often fatal disease African horse sickness in equids. The non-structural protein NS4 is the only AHSV protein that localizes to the nucleus. Here we report that all AHSV reference and representative field strains express one of the two forms of NS4, i.e. NS4-I or NS4-II. Both forms of NS4 are nucleocytoplasmic proteins, but NS4-I has a stronger nuclear presence whilst NS4-II has a proportionally higher cytoplasmic distribution. A subtype of NS4-II containing a nuclear localization signal (NLS), named NLS-NS4-II, displays distinct punctate foci in the nucleus. We showed that NS4 likely enters the nucleus via passive diffusion as a result of its small size. Colocalization analysis with nuclear compartments revealed that NS4 colocalizes with promyelocytic leukaemia nuclear bodies (PML-NBs), suggesting a role in the antiviral response or interferon signalling. Interestingly, we showed that two other AHSV proteins also interact with nuclear components. A small fraction of the NS1 tubules were present in the nucleus and associated with PML-NBs; this was more pronounced for a virus strain lacking NS4. A component of nuclear speckles, serine and arginine rich splicing factor 2 (SRSF2) was recruited to viral inclusion bodies (VIBs) in the cytoplasm of AHSV-infected cells and colocalized with NS2. Nuclear speckles are important sites for cellular mRNA transcript processing and maturation. Collectively, these results provide data on three AHSV non-structural proteins interacting with host cell nuclear components that could contribute to overcoming antiviral responses and creating conditions that will favour viral replication.

Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death.

Host nucleases are implicated in antiviral response through the processing of pathogen-derived nucleic acids. Among many host RNases, decapping enzymes DCP1 and 2, and 5'→3' exonuclease XRN1, which are components of the RNA decay machinery, have been extensively studied in prokaryotes, plants, and invertebrates but less so in mammalian systems. As a result, the implication of XRN1 and DCPs in viral replication, in particular, the spatio-temporal dynamics during RNA viral infections remains elusive. Here, we highlight that XRN1 and DCPs play a critical role in limiting several groups of RNA viral infections. This antiviral activity was not obvious in wild-type cells but clearly observed in type I interferon (IFN-I)-deficient cells. Mechanistically, infection with RNA viruses induced the enrichment of XRN1 and DCPs in viral replication complexes (vRCs), hence forming distinct cytoplasmic aggregates. These aggregates served as sites for direct interaction between XRN1, DCP1/2, and viral ribonucleoprotein that contains viral RNA (vRNA). Although these XRN1-DCP1/2-vRC-containing foci resemble antiviral stress granules (SGs) or P-body (PB), they did not colocalize with known SG markers and did not correlate with critical PB functions. Furthermore, the presence of 5' mono- and 5' triphosphate structures on vRNA was not required for the formation of XRN1-DCP1/2-vRC-containing foci. On the other hand, single-, double-stranded, and higher-ordered vRNA species play a role but are not deterministic for efficient formation of XRN1-DCP1/2 foci and consequent antiviral activity in a manner proportional to RNA length. These results highlight the mechanism behind the antiviral function of XRN1-DCP1/2 in RNA viral infections independent of IFN-I response, protein kinase R and PB function.

Measles Virus Bearing Measles Inclusion Body Encephalitis-Derived Fusion Protein Is Pathogenic after Infection via the Respiratory Route.

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host.

Solubilisation and purification of recombinant bluetongue virus VP7 expressed in a bacterial system.

Bluetongue virus (BTV) is an Orbivirus that has a profound economic impact due to direct loss of livestock as well as movement bans in an attempt to prevent the spread of the disease to susceptible areas. BTV VP7, along with VP3, forms the inner capsid core of the virus where it acts as the barrier between the outer layer and the inner core housing the genetic material. Purification of BTV VP7 has proven to be problematic and expensive mainly due to its insolubility is several expression systems. To overcome this, in this paper we present a protocol for the solubilisation of BTV VP7 from inclusion bodies expressed in E.coli, and subsequent purification using nickel affinity chromatography. The purified protein was then characterised using native PAGE, far ultraviolet circular dichroism (far-UV CD) and intrinsic fluorescence and found to have both secondary and tertiary structure even in the presence of 5 M urea. Both tertiary and secondary structure was further shown to be to be maintained at least to 42 °C in 5 M urea.

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.

Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence.

Promyelocytic leukemia nuclear bodies (PML-NB) are sub-nuclear organelles that are the hub of numerous proteins. DNA/RNA viruses often hijack the cellular factors resident in PML-NBs to promote their proliferation in host cells. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. The association of PML-NB proteins and HBV is being addressed in a number of recent studies. Here, we report that the PML-NB protein Speckled 110 kDa (Sp110) is SUMO1-modified and undergoes a deSUMOylation-driven release from the PML-NB in the presence of HBV. Intriguingly, Sp110 knockdown significantly reduced viral DNA load in the culture supernatant by activation of the type I interferon-response pathway. Furthermore, we found that Sp110 differentially regulates several direct target genes of hepatitis B virus protein X (HBx), a viral co-factor. Subsequently, we identified Sp110 as a novel interactor of HBx and found this association to be essential for the exit of Sp110 from the PML-NB during HBV infection and HBx recruitment on the promoter of these genes. HBx, in turn, modulates the recruitment of its associated transcription cofactors p300/HDAC1 to these co-regulated genes, thereby altering the host gene expression program in favor of viral persistence. Thus, we report a mechanism by which HBV can evade host immune response by hijacking the PML-NB protein Sp110, and therefore, we propose it to be a novel target for antiviral therapy.

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells.

Bacterial Inclusion Bodies (IBs) are amyloidal protein deposits that functionally mimic secretory granules from the endocrine system. When formed by therapeutically relevant proteins, they complement missing intracellular activities in jeopardized cell cultures, offering an intriguing platform for protein drug delivery in substitutive therapies. Despite the therapeutic potential of IBs, their capability to interact with eukaryotic cells, cross the cell membrane and release their functional building blocks into the cytosolic space remains essentially unexplored. We have systematically dissected the process by which bacterial amyloids interact with mammalian cells. An early and tight cell membrane anchorage of IBs is followed by cellular uptake of single or grouped IBs of variable sizes by macropinocytosis. Although an important fraction of the penetrating particles is led to lysosomal degradation, biologically significant amounts of protein are released into the cytosol. In addition, our data suggest the involvement of the bacterial cell folding modulator DnaK in the release of functional proteins from these amyloidal reservoirs. The mechanisms supporting the internalization of disintegrable protein nanoparticles revealed here offer clues to implement novel approaches for protein drug delivery based on controlled protein packaging as bacterial IBs.

Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies.

Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.

Efficient production of Tymovirus like particles displaying immunodominant epitopes of Japanese Encephalitis Virus envelope protein.

Japanese Encephalitis (JE) is a mosquito borne arboviral infection caused by Japanese Encephalitis Virus (JEV). It is a major cause of viral encephalitis in Asian countries including India. In the present study, we have used a Tymovirus [i.e. Physalis Mottle Virus (PhMV) coat protein (CP)], which forms virus like particles (VLPs) as a template to display immunodominant epitopes of JEV envelope (E) protein. The immunodominant epitopes of JEV were inserted at the N-terminus of the wild type PhMV CP, and these constructs were cloned and expressed in Escherichia coli. The chimeric proteins were purified from the inclusion bodies and evaluated for VLP formation. The purified protein was identified by Western blotting and VLP formation was studied and confirmed by transmission electron microscopy and dynamic light scattering. Finally, the immunogenicity was studied in mice. Our results indicate that the chimeric protein with JEV epitopes assembled efficiently to form VLPs generating neutralizing antibodies. Hence, we report the purified chimeric VLP would be a potent vaccine candidate, which needs to be evaluated in a mouse challenge model.

Viperin protein expression inhibits the late stage of respiratory syncytial virus morphogenesis.

We examined the effect of respiratory syncytial virus (RSV) infection on viperin protein expression in the permissive HEp2 and non-permissive RAW 264.7 macrophage cell lines. In RSV-infected HEp2 cells low levels of the viperin protein was localized to the virus-induced inclusion bodies and did not impair virus transmission in these cells. In contrast, RSV-infected RAW 264.7 cells increased expression of the STAT1 protein occurred at between 6 and 12h post-infection, which coincided with the appearance of P-STAT1. A relatively high level of viperin protein expression was detected in infected RAW 264.7 cells, and it was extensively localized throughout the cytoplasm of infected cells. The effect of early viperin protein expression on RSV infection in cells that are normally permissive to RSV cultivation was examined by using either transient transfected HEp2 cells or stable transfected HeLa cells that expressed the viperin protein. The early expression of viperin in HeLa cells did not prevent virus infection, and no significant inhibitory effect on either virus protein expression or targeting of virus proteins to the cell surface was noted. However, while inclusion body formation was not inhibited, early viperin protein expression was associated with the inhibition of virus filament formation and reduced cell-to-cell virus transmission. Inhibition of virus filament formation was also observed in HEp2 cells expressing viperin. Collectively our data suggested that viperin impaired RSV transmission by inhibiting virus filament formation, providing a basis for its anti-virus activity in RSV-infected cells.

Mutations within A 35 amino acid region of P6 influence self-association, inclusion body formation, and Caulimovirus infectivity.

Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1-D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection.

Potato virus Y HCPro localization at distinct, dynamically related and environment-influenced structures in the cell cytoplasm.

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.

Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins.

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.