Abstract: Microtubules, components of the cell cytoskeleton, play a central role in cellular trafficking. Here we show that rotavirus infection leads to a remodeling of the microtubule network together with the formation of tubulin granules. While most microtubules surrounding the nucleus depolymerize, others appear packed at the cell periphery. In microtubule depolymerization areas, tubulin granules are observed; they colocalize with viroplasms, viral compartments formed by interactions between rotavirus proteins NSP2 and NSP5. With purified proteins, we show that tubulin directly interacts in vitro with NSP2 but not with NSP5. The binding of NSP2 to tubulin is independent of its phosphatase activity. The comparison of three-dimensional (3-D) reconstructions of NSP2 octamers alone or associated with tubulin reveals electron densities in the positively charged grooves of NSP2 that we attribute to tubulin. Site-directed mutagenesis of NSP2 and competition assays between RNA and tubulin for NSP2 binding confirm that tubulin binds to these charged grooves of NSP2. Although the tubulin position within NSP2 grooves cannot be precisely determined, the tubulin C-terminal H12 alpha-helix could be involved in the interaction. NSP2 overexpression and rotavirus infection produce similar effects on the microtubule network. NSP2 depolymerizes microtubules and leads to tubulin granule formation. Our results demonstrate that tubulin is a viroplasm component and reveal an original mechanism. Tubulin sequestration by NSP2 induces microtubule depolymerization. This depolymerization probably reroutes the cell machinery by inhibiting trafficking and functions potentially involved in defenses to viral infections.
Abstract: Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5' end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5' untranslated region (5'-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5'-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.
Abstract: Rotavirus-like particles (VLPs) have shown promise as rotavirus vaccine candidates in mice, rabbits and pigs. In pigs, VLP vaccines reduced rotavirus shedding and disease but only when used in conjunction with live attenuated human rotavirus. Using a porcine rotavirus pig model, rotavirus antigen shedding was reduced by up to 40% after vaccination with VLPs including the neutralizing antigens VP7 and VP8* when used in combination with the adjuvant polyphosphazene poly[di(carbozylatophenoxy)phoshazene] (PCPP). In contrast, complete protection from rotavirus antigen shedding and disease was induced by vaccination with the virulent porcine rotavirus PRV 4F. This is the first study to demonstrate some post-challenge reductions in rotavirus antigen shedding in a pig model of rotavirus disease after vaccination with VLPs without combining with infectious rotavirus.
Abstract: Among Caliciviridae, the norovirus genus encompasses enteric viruses that infect humans as well as several animal species, causing gastroenteritis. Porcine strains are classified together with human strains within genogroup II, whilst bovine norovirus strains represent genogroup III. Various GI and GII human strains bind to carbohydrates of the histo-blood group family which may be shared among mammalian species. Genetic relatedness of human and animal strains as well as the presence of potentially shared ligands raises the possibility of norovirus cross-species transmission. In the present study, we identified a carbohydrate ligand for the prototype bovine norovirus strain Bo/Newbury2/76/UK (NB2). Attachment of virus-like particles (VLPs) of the NB2 strain to bovine gut tissue sections showed a complete match with the staining by reagents recognizing the Galalpha1,3 motif. Alpha-galactosidase treatment confirmed involvement of a terminal alpha-linked galactose. Specific binding of VLPs to the alphaGal epitope (Galalpha3Galbeta4GlcNAcbeta-R) was observed. The binding of Galalpha3GalalphaOMe to rNB2 VLPs was characterized at atomic resolution employing saturation transfer difference (STD) NMR experiments. Transfection of human cells with an alpha1,3galactosyltransferase cDNA allowed binding of NB2 VLPs, whilst inversely, attachment to porcine vascular endothelial cells was lost when the cells originated from an alpha1,3galactosyltransferase KO animal. The alphaGal epitope is expressed in all mammalian species with the exception of the Hominidaea family due to the inactivation of the alpha1,3galactosyltransferase gene (GGTA1). Accordingly, the NB2 carbohydrate ligand is absent from human tissues. Although expressed on porcine vascular endothelial cells, we observed that unlike in cows, it is not present on gut epithelial cells, suggesting that neither man nor pig could be infected by the NB2 bovine strain.
Abstract: The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.
Abstract: In presence of low or high levels of rotavirus-specific maternal antibodies, the ability of newborn mice to respond to immunization with rotavirus RF 8*-2/6/7 VLPs, was evaluated. After parenteral vaccination, 100% of offspring born to low-antibody-titer dams developed rotavirus-specific IgG antibodies (n=7). In contrast, only 25% of offsprings born to high-antibody-titer dams responded to parenteral immunization (n=12). When comparing parenteral versus oral immunization in offspring to low-antibody-titer dams only 45% responded after oral immunization (n=6). In conclusion, the response to parenteral immunization was not hampered by the presence of low levels of maternal antibodies induced by a natural infection while oral immunization was impaired. However, high levels of maternal antibodies impaired the response to parenteral immunization.
Abstract: Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T = 13 to T = 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Abstract: Rotavirus virus-like particles (RV-VLPs) represent a novel strategy for development of a rotavirus subunit vaccine. In this study, RF 8-2/6/7-VLPs with rotavirus VP8 protein (amino acid 1-241 of VP4) fused to the amino terminal end of a truncated VP2, were evaluated for their immunogenic and protective properties. A single intramuscular dose of, either 2 or 20 microg, RF 8-2/6/7-VLPs alone or combined with a potent adjuvant poly[di(carboxylatophenoxy)]phosphazene] (PCPP) induced rotavirus-specific serum IgG and IgA, fecal IgG titers that were enhanced 5-90-fold by adjuvant. Passive protective immunity was achieved in offspring to dams vaccinated with 2 and 20 microg RV-VLPs in presence of adjuvant and 20 microg RV-VLP without adjuvant.
Abstract: Rotavirus nonstructural protein NSP3 interacts specifically with the 3' end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.
Abstract: Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.
Abstract: Dendritic cells (DC) represent the link between innate and adaptive immunity. They are classified as antigen-presenting cells (APC) and can initiate and modulate the immune response. To investigate the interaction with DCs, live RF-81 bovine rotavirus strain (RFV) and rotavirus-like particles (rota-VLP), RF 2/6-GFP-VLP and rota RF 8*2/6/7-VLP, were added in vitro to murine bone marrow-derived DCs (bmDCs). Live RFV, RF 2/6-GFP-VLP and RF 8*2/6/7-VLP all bound to bmDC and were internalized but only live RFV stimulated phenotypic maturation of the bmDCs as shown by the upregulation of the co-stimulatory molecule CD86. Even though bmDCs internalized RF 2/6-GFP-VLP and RF 8*2/6/7-VLP as efficiently as live RFV, these rota-VLP were not able to activate the cells. Supernatants derived from bmDC cultures treated with live RFV, RF 2/6-GFP-VLP or RF 8*2/6/7-VLP were examined for TNF-alpha production. At 6, 18 and 24 h post-infection, TNF-alpha concentrations were significantly increased in cultures treated with live RFV and rota-VLP compared with untreated cultures. In conclusion, this study showed that live RF-81 bovine rotavirus strain was internalized and induced bmDCs activation, whereas both RF 2/6-GFP-VLP and RF 8*2/6/7-VLP were internalized by bmDCs without triggering their activation.
Abstract: To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.
Abstract: The rotavirus capsid is made up of three concentric protein layers. The outer layer, consisting of VP7 and VP4, is lost during virus entry into the host cell. Rotavirus field isolates can be adapted to high-titre growth in tissue culture by treatment with trypsin and by supplementing the culture medium with trypsin, which cleaves VP4 into two fragments, VP8* and VP5*. It is known that protease inhibitors reduce the replication of rotavirus in vitro and in vivo and also diminish disease symptoms in a mouse model. To clarify the molecular basis of these observations, a series of assays were conducted on purified rotavirus particles grown in the presence of trypsin. Results of HPLC and mass spectrometry followed by N-terminal sequencing showed that viral particles contain molecules of trypsin. When associated with triple-layer particles (TLPs), trypsin is inactive and not accessible to protease inhibitors, such as aprotinin. When the outer layer is solubilized by calcium-chelating agents, VP5*, VP8* and VP7 are released and the associated trypsin is activated, allowing cleavage of the viral capsid proteins, as well as other exogenous proteins. It is shown that addition of trypsin inhibitors significantly reduces synthesis of viral mRNA and viral proteins in cells and has a major inhibitory effect if present when virus enters the cell. These data indicate that incorporation of trypsin into rotavirus particles may enhance its infectivity.
Abstract: Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3' consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.
Abstract: Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.
Abstract: A human rotavirus (isolate M) with an atypical electropherotype with 14 apparent bands of double-stranded RNA was isolated from a chronically infected immunodeficient child. MA-104 cell culture adaptation showed that the M isolate was a mixture of viruses containing standard genes (M0) or rearranged genes: M1 (containing a rearranged gene 7) and M2 (containing rearranged genes 7 and 11). The rearranged gene 7 of virus M1 (gene 7R) was very unusual because it contained two complete open reading frames (ORF). Moreover, serial propagation of virus M1 in cell culture indicated that gene 7R rapidly evolved, leading to a virus with a deleted gene 7R (gene 7RDelta). Gene 7RDelta coded for a modified NSP3 protein (NSP3m) of 599 amino acids (aa) containing a repetition of aa 8 to 296. The virus M3 (containing gene 7RDelta) was not defective in cell culture and actually produced NSP3m. The rearranged gene 11 (gene 11R) had a more usual pattern, with a partial duplication leading to a normal ORF followed by a long 3' untranslated region. The rearrangement in gene 11R was almost identical to some of those previously described, suggesting that there is a hot spot for gene rearrangements at a specific location on the sequence. It has been suggested that in some cases the existence of short direct repeats could favor the occurrence of rearrangement at a specific site. The computer modeling of gene 7 and 11 mRNAs led us to propose a new mechanism for gene rearrangements in which secondary structures, besides short direct repeats, might facilitate and direct the transfer of the RNA polymerase from the 5' to the 3' end of the plus-strand RNA template during the replication step.
Abstract: Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.
Abstract: Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.
Abstract: In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3'-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3' end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.
Abstract: The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.
Abstract: The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.
Abstract: The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.
Abstract: Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.
Abstract: NSP5 (NS26), the product of rotavirus gene 11, is a phosphoprotein whose role in the virus replication cycle is unknown. To gain further insight into its function, we obtained monoclonal antibodies against the baculovirus-expressed protein. By immunoprecipitation and immunoblotting experiments, we showed that (i) NSP5 appears in many different phosphorylated forms in rotavirus-infected cells; (ii) immunoprecipitated NSP5 from rotavirus-infected cells can be phosphorylated in vitro by incubation with ATP; (iii) NSP5, produced either by transient transfection of rotavirus gene 11 or by infection by gene 11 recombinant vaccinia virus or baculovirus, can be phosphorylated in vivo and in vitro; (iv) NSP5 expressed in Escherichia coli is phosphorylated in vitro, and thus NSP5 is a potential protein kinase; and (v) NSP5 forms dimers and interacts with NSP2. The intracellular localization of NSP5 in the course of rotavirus infection and after transient expression in COS7 cells has also been investigated. In rotavirus-infected cells, NSP5 is localized in viroplasms, but it is widespread throughout the cytoplasm of transfected COS7 cells. NSP5 produced by transfected COS7 cells did not acquire the multiphosphorylated forms observed in rotavirus-infected COS7 cells. Thus, there is a tight correlation between the localization of NSP5 in the viroplasms and its protein kinase activity in vivo or in vitro. Our results suggest that cellular or viral cofactors are indispensable to fully phosphorylate NSP5 and to reach its intracellular localization.
Abstract: Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7.
Abstract: The genomes of viruses in the family Reoviridae consist of segmented double-stranded RNA. There are 10 to 12 segments depending on the genus. The 5' ends and the 3' ends of the RNAs present conserved motifs for each virus genus. These conserved motifs have been hypothesized to play a role in genomic segment assortment during virus morphogenesis. Using a set of monoclonal antibodies we have tried to identify rotaviral proteins that bind to RNA during infection in cell culture. This methodology takes advantage of being able to label RNA in vitro to high specific activity and also of solid phase processing of RNA-protein complexes. After cross-linking the RNA to protein in infected cells, protein-RNA complexes are precipitated with a specific MAb; then, the RNA in the complex is labeled in vitro and the protein or nucleic acid moieties are analyzed by usual protocols. This paper describes results using an anti NSP3 MAb. In infected cells, we have shown that NSP3 binds to the eleven messenger RNAs, and that a sequence from nucleotides 8 to 15 is protected from digestion with RNAse T1 by NSP3 in the RNA-protein complex. The availability of recombinant protein NSP3 expressed in the baculovirus-insect cell system has allowed the sequence specificity of NSP3 to be studied in vitro. The minimal sequence recognized by NSP3 is GACC. The role of NSP3 in rotavirus replication is discussed based on these results and by comparison with other RNA-binding proteins of members of the Reoviridae family.
Abstract: Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.
Abstract: The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.
Abstract: The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.
Abstract: Interaction between viral proteins and RNAs has been studied in rotavirus-infected cells. The use of UV cross-linking followed by immunoprecipitation and labeling with T4 polynucleotide kinase allowed us to detect interactions between RNA and nonstructural viral proteins. The RNAs linked to the nonstructural protein NSP3 have been identified as rotavirus mRNAs, and the sequences of the RNase T1-protected fragments have been established. These sequences correspond to the 3' end sequence common to all rotavirus group A genes. We also show that the last 3' nucleotide is cross-linked to the protein and that monomeric and multimeric forms of NSP3 are bound to rotavirus mRNA. The role of NSP3 in rotavirus replication is discussed in the light of our results and by comparison with other RNA-binding proteins of members of the Reoviridae family.
Abstract: C57BL/6 (H-2b) mice were primed with the bovine RF strain of rotavirus to study the induction of CD8+ cytotoxic T lymphocytes (CTLs). These rotavirus-specific CTLs were detected only after in vitro restimulation with the virus. Using a recombinant vaccinia virus we identified the RF VP7 protein as a major target of these CTLs. The response against this protein was obtained also after in vitro restimulation with simian SA11 and human WA strains of rotavirus. Using published Db and Kb allele-specific motifs to predict possible CTL epitopes in the RF VP7 protein, we synthesized and tested 18 predicted peptides of VP7. Only one peptide was able to sensitize target cells at a concentration below 5 x 10(-7) M. This CTL epitope was also induced by immunization with the RF VP7 expressed with a baculovirus vector, and was shown to be immunodominant by its capacity to inhibit, in an unlabelled target assay, the bulk response against cells infected with recombinant vaccinia virus expressing VP7. This CTL epitope overlaps the H2 signal peptide of the protein.
Abstract: To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.
Abstract: cDNA molecules encoding the major structural protein (VP6) of the Simian rotavirus SA11 were inserted under the control of the vaccinia virus 7.5 kDa promoter into the thymidine kinase gene. Synthesis of VP6 was demonstrated by immunoprecipitation of recombinant virus-infected cell. Mice inoculated via several routes with this recombinant vaccinia produce high titers of antirotavirus antibodies lacking neutralizing activity.
Abstract: Glycosylation and translocation of the simian rotavirus protein VP7, a resident ER protein, does not occur co-translationally in vivo. In pulse-chase experiments in COS cells, nonglycosylated VP7 was still detectable after a 25-min chase period, although the single glycosylation site was only 18 residues beyond the signal peptide cleavage site. After labeling, glycosylated and nonglycosylated VP7 was recovered in microsomes but the latter was sensitive to trypsin (i.e., the nascent protein became membrane associated) but most of it entered the ER posttranslationally because of a rate-limiting step early in translocation. In contrast with the simian protein, bovine VP7 was glycosylated and translocated rapidly. Thus, delayed translocation per se was not required for retention of VP7 in the ER. By constructing hybrid proteins, it was further shown that the signal peptide together with residues 64-111 of the simian protein caused delayed translocation. The same sequences were also necessary and sufficient for retention of simian VP7 in the ER. The data are consistent with the idea that certain proteins are inserted into the ER membrane in a loop configuration.
Abstract: We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.
Abstract: A plaque hybridization assay was adapted to rotavirus. In this test cDNA or oligonucleotide probes were used to discriminate between plaques originating from virus carrying genes of bovine and simian origin. Only mRNAs present in infected cells were detected as demonstrated by using oligonucleotides corresponding to both strands. This assay can be used to screen reassortants or mutants of rotaviruses.
Abstract: During the study of the DNA from 25 beta-thalassemic subjects from mediterranean origin the polymorphic Taq I restriction site located 3-kb 5' to the human delta-globin gene, was found non-randomly associated to the polymorphic Hind III sites within the G gamma- and A gamma-globin genes. This indicates that the 3' limit of the linkage group of polymorphic restriction sites including the gamma-globin genes is located downstream to the polymorphic Taq I site.
Abstract: Several reports have suggested but not proven that the large intervening sequence of Lepore delta-beta fusion gene was of beta-type (3-5). A method able to detect rearrangements as small as 4 nucleotide pairs directly into genomic DNA (6) has been applied to the total DNA of a heterozygous Lepore-Boston patient in order to identify accurately the origin of the large intervening sequence of the delta-beta fusion-gene. Hybrid duplexes were formed between genomic Lepore DNA and single-stranded DNA used as probes, then submitted to S1-nuclease treatment. Our data demonstrate that the entire large intervening sequence of the Lepore fusion gene is of beta-type. Moreover, no large modification was detected in any delta- and beta parts of the delta-beta fusion gene.
Abstract: Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].
Abstract: Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster. Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated. Thus, any recognized "CG enzyme" site can be detected since they are unmethylated. Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites. The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system. Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region. In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region. Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene. The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively.
