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.

Investigating the role of Ebp1 in Chandipura virus infection.

ErbB-3 binding protein 1 (Ebp1) is a host protein which binds ErbB-3 receptor to induce signalling events for cell growth regulation. In addition, Ebp1 also interacts with ribonucleoprotein complexes. In recent times, Ebp1 was found to play an antagonistic role in viral infections caused by Influenza and Rinderpest viruses. In our present work we have tried to understand the role of Ebp1 in Chandipura virus (CHPV) infection. We have observed an induction in Ebp1 expression upon CHPV infection similar to other viruses. However, unlike other viruses an overexpressed Ebp1 only reduces viral protein expression, but does not affect its progeny formation. Additionally, this effect is being carried out in an indirect manner, as there is no interaction between Ebp1 and viral proteins. This is despite Ebp1's presence in viral inclusion bodies.

The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells.

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.

Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

BACKGROUND: Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). METHODS: Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. RESULTS: The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). CONCLUSION: We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18.

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.

Focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein P.

UNLABELLED: The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE: Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.

Crystal lattice as biological phenotype for insect viruses.

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.

Avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of sigmaNS and lambdaA to microNS inclusions.

We have recently shown that the avian reovirus non-structural protein microNS forms cytoplasmic inclusions in transfected cells and recruits sigmaNS to these structures. In the present study we further demonstrate that microNS mediates the association of the major core protein lambdaA, but not of sigmaA or sigmaC, with inclusions, indicating that the recruitment of viral proteins into avian reovirus factories has specificity. Thus, some proteins appear to be initially recruited to factories by association with microNS, whereas others are recruited subsequently through interaction with as-yet-unknown factors. We next used metabolic pulse-chase radiolabeling combined with cell fractionation and antibody immunoprecipitation to study the recruitment of newly synthesized viral polypeptides into viral factories and virus particles. The results of this combined approach revealed that avian reovirus morphogenesis is a complex and temporally controlled process that takes place exclusively within globular viral factories that are not microtubule-associated. Our findings further suggest that cores are assembled within the first 30 minutes after the synthesis of their polypeptide components, and that reovirion morphogenesis is completed over the next 30 minutes by the subsequent addition of outer capsid proteins.

Avian reovirus nonstructural protein microNS forms viroplasm-like inclusions and recruits protein sigmaNS to these structures.

The M3 genome segment of avian reovirus 1733, which encodes the nonstructural protein microNS, is 1996 nucleotides long and contains a long open reading frame that is predicted to encode a polypeptide of 635 amino acid residues. Examination of the deduced amino acid sequence of microNS revealed the presence of two regions near its carboxyl terminus with a high probability of forming alpha-helical coiled coils. Expression of the M3 gene in both infected and transfected cells revealed that this gene specifies two protein isoforms that are recognized by a microNS-specific antiserum. Only the larger microNS isoform, but not the smaller one, interacts with the nonstructural protein sigmaNS in infected cells, suggesting that the two isoforms play different roles during avian reovirus infection. In the second part of this study, we show that microNS and the nonstructural protein sigmaNS colocalize throughout the viral life cycle in large and small phase-dense globular cytoplasmic inclusions, which are believed to be the sites of viral replication and assembly. Individual expression of these proteins in transfected cells of avian and mammalian origin revealed that while microNS is able to form inclusions in the absence of other viral proteins, sigmaNS distributes diffusely throughout the cytoplasm in the absence of microNS. These data suggest that microNS is the minimal viral factor required for inclusion formation during avian reovirus infection. On the other hand, our findings that sigmaNS associates with microNS in infected cells, and that sigmaNS colocalizes with microNS in viroplasm-like inclusions when the two proteins are coexpressed in transfected cells, suggest that microNS mediates the association of sigmaNS to inclusions in avian reovirus-infected cells.

Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy.

Infection of the cerebral cortical neurons with JC virus (JCV) with possible dysplastic ganglion-like alteration of the infected neurons found in a case of progressive multifocal leukoencephalopathy (PML) is described. The patient was a 21-year-old man with common variable immunodeficiency who died of PML after a 9-month clinical course. At autopsy, the white matter of the cerebrum, brainstem, cerebellum, and spinal cord exhibited extensive demyelination and necrosis. Numerous inclusion-bearing oligodendrocytes and bizarre astrocytes were found. In the occipital and temporal cortex, thick band-like aggregates of dysplastic ganglion-like cells (DGLCs) were found. These DGLCs showed immunohistochemical properties of neurons, and nuclei of some DGLCs were immunoreactive for large T antigen of SV40/JCV and p53, but not for capsid protein JCV VP1. In situ hybridization for mRNA of JCV large T antigen revealed positive signals in the nuclei of some DGLCs. These results indicate that JCV infected neurons and it is suggested that binding of the large T antigen with cellular proteins could have resulted in the dysplastic, ganglion cell-like change of the infected neurons, although the possibility that the aggregates of DGLCs represent a pre-existent malformative lesion of the cortex cannot be excluded completely.

Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus.

Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections. Antiserum to CP reacted with virions of seven out of 18 other potyviruses. CP was distributed throughout the cytoplasm of infected cells. Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses. NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions. Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein. Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP. An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus. Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated. Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI. Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments. The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP.

Diagnosis of avian adenovirus infections using DNA in situ hybridization.

Three DNA oligonucleotide probes designated FN-23, FN-48, and FN-96 were evaluated for the diagnosis of aviadenovirus infections by DNA in situ hybridization. Paraffin-embedded tissues were obtained from birds with confirmed adenovirus infection, birds with putative adenovirus infections, and birds with intranuclear inclusions caused by herpesvirus and polyomavirus. In birds with confirmed adenovirus infection, probes FN-23 and FN-96 identified 78% and 72% of diseased individuals, respectively. Only probe FN-48 detected chickens with group II adenovirus infection. In birds with putative adenovirus infection, the DNA probes confirmed aviadenovirus infections in 76% of the population. Probes FN-23, FN-96, and FN-48 detected 85%, 74%, and 18% of adenovirus-infected birds, respectively. None of the DNA probes cross-hybridized with tissues from polyomavirus-infected psittaciform birds or with tissues from a chicken with infectious laryngotracheitis. In contrast, probe FN-23 did cross-hybridize to herpesvirus-infected tissues from two of eight psittaciform birds with Pacheco's parrot disease. Probes FN-48 and FN-96 did not react with these tissues.

Sequestration of PML and Sp100 proteins in an intranuclear viral structure during herpes simplex virus type 1 infection.

We investigated the intranuclear distribution of PML and Sp100 in HeLa cells at the ultrastructural level and examined their relocalization in response to herpes simplex virus type 1 (HSV-1) infection. In the absence of infection, we observed that both are components, not only of nuclear bodies, but also of interchromatin granule-associated zones, which suggests a potential role for PML and Sp100 in splicing events. Prolonged HSV-1 infection induced dramatic changes in nuclear organization which consisted of the morphological disappearance of some nuclear structures (nuclear bodies, interchromatin granule-associated zones, coiled bodies) and of the development of a centrally located electron-translucent viral region which pushed the cellular clusters of interchromatin granules to the nuclear border. Concomitantly, dense bodies, concentric arrays of reduplicated inner nuclear membrane, and translucent patches containing a few viral capsids occurred at the nuclear border. PML and Sp100 were exclusively detected over the finely granular material of the viral translucent patches which also contains small amounts of p80-coilin and U1 and U2 snRNAs. An antiserum raised against capsid proteins intensely labeled the viral translucent patches at the level of their finely granular material and enclosed viral capsids. Our data, therefore, suggest that these viral structures, in addition to being the site of accumulation of viral capsid proteins and, possibly, a capsidworks, are also a site of sequestration of cell factors including PML and Sp100. Viral capsid proteins could interfere with and inactivate PML and Sp100 and be implicated in the shutoff of host cell metabolism induced by HSV-1 infection.

Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains.

The failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorhamnosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native-like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.

The Melolontha melolontha entomopoxvirus (MmEPV) fusolin is related to the fusolins of lepidopteran EPVs and to the 37K baculovirus glycoprotein.

We have cloned and sequenced a 1.7-kbp DNA fragment of the MmEPV genome encompassing the major polypeptide of the spindle-shaped inclusions gene termed fusolin. The sequence contained a single open reading frame of 1203 nt capable of coding for a polypeptide of 45.8 kDa. The 13 N-terminal amino acid (aa) residues were hydrophobic and could act as a signal peptide. The aa sequence also contained 13 cysteine residues very likely involved in paracrystal formation. This sequence showed significant homologies with the fusolins of two lepidopteran EPVs, the Choristoneura biennis EPV (CbEPV) and the Heliothis armigera EPV, and also with the 37K glycoproteins of Autographa californica and Orgyia pseudotsugata baculoviruses. No homology was found between the MmEPV fusolin and the 100K MmEPV spherulin, nor with the 110K polypeptide of the CbEPV and Amsacta moorei EPV spheroidins. These data were confirmed by Western blot analysis. Transfection of vaccinia-infected mammalian cells with a plasmid encompassing the fusolin sequence plus the upstream regulatory region resulted in transient expression of the gene. This indicated that the vaccinia transcription machinery is able to transcribe the fusolin gene. The fusolin was also expressed in insect cells via a recombinant baculovirus.

Expression of early and late adenoviral proteins in fatal adenovirus bronchopneumonia.

The localization and distribution of three adenoviral proteins, hexon, E1A, and 55-kDa E1B, in 16 cases of fatal adenovirus bronchopneumonia in infants and children, are described. The proteins were immunohistochemically demonstrated in paraffin sections using monoclonal antibodies followed by the avidin-biotin-peroxidase method. The hexon antigen was present in inclusion-bearing bronchial, bronchiolar, and alveolar cells, mainly in the so-called rosette cells, as well as in necrotic debris in necrotizing areas. E1A antigen was also recognized in cells with nuclear inclusions where the reaction decorated the inclusion, nuclear chromatin, and cytoplasm but distributed mainly in alveolar cells and to a lesser extent in bronchial and bronchiolar cells. The 55-kDa E1B protein was extensively present in "activated," reactive-appearing, nuclei of bronchial, bronchiolar, and alveolar epithelial cells and in the cytoplasm of rare cells having nuclear inclusions. These activated nuclei did not stain for the other two antigens. "Smudge" cells reacted poorly or not at all with any of the antibodies. The reactivity found produced a sort of complementary pattern between the hexon-positive, inclusion-containing cells and the 55-kDa E1B-positive, inclusion-noncontaining cells. The relationships of present findings and virologic data are discussed.

Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein.

The open reading frame 2 (ORF2) of the potexviral genome encodes a 24- to 26-kDa protein which is part of the "triple gene block," a group of overlapping ORFs also present in the genomes of the carla-, hordei-, and furoviruses. The product of these ORFs is believed to play a role in the cell-to-cell movement of the viruses in host plants. The amino acid sequences of the homologous ORF2 products encoded by these related viruses suggest that they specify NTP binding and possibly helicase activities. We have used an Escherichia coli expression system to produce significant amounts of the 26-kDa protein (p26) encoded by foxtail mosaic potexvirus ORF2. p28 was purified to near homogeneity by conventional purification methods and some of its biochemical properties were determined. We present evidence that p26 is an ATP, CTP, and RNA binding protein with apparent ATPase activity. Western blot analysis of infected plant extracts using a polyclonal antiserum produced against p26 indicates that it is a relatively stable protein maintained at high levels for at least 6 days following its peak level of expression. Moreover, it is found predominantly in the soluble fraction of infected tissues. An immunocytochemical analysis of infected Chenopodium quinoa leaves reveals that p26 is exclusively associated with cytoplasmic inclusions in proximity to but distinct from aggregates of viral particles.

Purification and characterization of the U-particle, a cellular constituent whose synthesis is stimulated by Mengovirus infection.

We have isolated a cellular protein particle whose synthesis is induced by infection with Mengovirus or TMEV. The U-particle inhibits translation in vitro and binds to both capped and uncapped mRNA's. It is spherical, 12 nm in diameter, and is composed of multiple copies of two polypeptide subunits having molecular weights of 23,000 and 25,000 which do not appear to be glycosylated or phosphorylated. U-particles are capable of inhibiting mRNA translation in vitro.

The cellular U-particle, whose synthesis is induced by mengovirus infection, is homologous to apoferritin.

Mengo virus infection of mouse L-cells results in induction of the synthesis of a cellular protein-containing particle, 12 nm in diameter, which was designated U (Boege et al. (1987) Virology 159, 358-367). We have purified the U-particle from virus-infected cells by a series of chromatographic steps and found it to be composed of two polypeptide species (MW 23,000 and 25,000), present in a ratio of approximately 7:3. Neither of these polypeptides is measurably glycosylated or phosphorylated and the U-particle contains no detectable nucleic acid. Several amino acid sequences obtained from CNBr fragments of the U-polypeptides identified them as the H- and L-chains of mouse apoferritin. This finding was supported by immunoblotting and electron microscopy. In terms of function, the U-particle/apoferritin effectively inhibits the translation of mRNAs in reticulocyte lysates. These experiments indicate that apoferritin may perform important functions in eukaryotic cells in addition to iron storage. Finally, we propose mechanisms to explain how Mengo virus infection could specifically induce the synthesis of apoferritin and how increasing amounts of cytoplasmic apoferritin could facilitate virus replication.

Characterization of virus inclusion bodies in bluetongue virus-infected cells.

A combined qualitative and quantitative approach has been used to examine the role of virus inclusion bodies (VIBs) in the morphogenesis of bluetongue virus (BTV). VIBs were detected as early as 4 h post-infection (p.i.), and their number and profile areas increased significantly between 12 and 16 h, and 20 and 28 h p.i. respectively. Core- and virus-like particles were found within and at the periphery of the VIB matrix, respectively, and their numerical density (number per area of VIB matrix) decreased during the course of infection whereas the numerical density of virus particles in the cytoplasm increased. Virus-like particles had a diameter of 57 +/- 8 nm and core-like particles appeared to fall into two size ranges, 32 +/- 3 nm and 38 +/- 3 nm in diameter. Both pre- and post-embedding immunoelectron microscopy procedures were used to localize BTV structural and non-structural proteins within the VIBs. The VIB matrix was labelled with antibodies to structural proteins VP5 and VP7 and non-structural proteins NS1 and NS2. Cores within VIBs contained proteins VP5, VP7 and NS1 but not VP2. Virus-like particles at the periphery of VIBs contained VP2, VP5, VP7 and NS1. The results suggest that BTV particles are synthesized, assembled and released from the perimeter of VIBs and not from within the matrix. Cores embedded in the VIBs are likely to have been trapped there during expansion of the matrix during replication.