Effects of viral protein, viral RNA, and some other polyelectrolytes on infection of tobacco protoplasts by TMV.
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Caseins↗
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Large language models improve annotation of prokaryotic viral proteins.
Viral genomes are poorly annotated in metagenomic samples, representing an obstacle to understanding viral diversity and function. Current annotation approaches rely on alignment-based sequence homology methods, which are limited by the paucity of characterized viral proteins and divergence among viral sequences. Here we show that protein language models can capture prokaryotic viral protein function, enabling new portions of viral sequence space to be assigned biologically meaningful labels. When applied to global ocean virome data, our classifier expanded the annotated fraction of viral protein families by 29%. Among previously unannotated sequences, we highlight the identification of an integrase defining a mobile element in marine picocyanobacteria and a capsid protein that anchors globally widespread viral elements. Furthermore, improved high-level functional annotation provides a means to characterize similarities in genomic organization among diverse viral sequences. Protein language models thus enhance remote homology detection of viral proteins, serving as a useful complement to existing approaches.
Viral Proteins↗
Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.
In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing 1a and 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5',3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable of viral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.
Bromovirus↗
Human parainfluenza virus 3: purification and characterization of subviral components, viral proteins and viral RNA.
A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18-1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180 000; P, 83 000; HN, 69 000; NP, 66 000; F0, 60 000; F1, 51 000; and M, 38 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes F0 and F1). F1.2, the activated, cleaved, fusion glycoprotein (60 000 Da), consisting of two disulfide-linked subunits, F1 and F2, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F2 could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP. Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29-1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 X 10(6).
Animals↗
[Iclusion proteins and viral proteins of the baculovirus of the dipteran Tipula paludosa (Meigen)].
Examination of the polyhedron protein by polyacrylamide gel electrophoresis shows only one polypeptide with a molecular weight of 25,500 +/- 500 daltons, while that of virion proteins reveals 13 polypeptides. No antigenic community could be demonstrated between the polyhedron protein of the Baculovirus of T. paludosa and the polyhedron protein of several other Baculoviruses.
Animals↗
Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease.
In recent years, infectious bursal disease virus (IBDV) has become a serious economic problem as a result of the emergence of new and very virulent strains. Most of the antibodies produced against IBDV are for the structural proteins viral protein (VP) 2 (VP2) and VP3. The purpose of this study was to test the potential of recombinant VP3 to induce protective antibodies. The gene for VP3 was isolated from a virulent strain of the virus and cloned into prokaryotic (Escherichia coli) and eukaryotic (baculovirus) expression systems. The protein expressed by both systems was of the expected size (32 kD) and was detected by anti-IBDV antibodies. Following partial purification, the polypeptides were injected into intact birds and induced the production of high levels of anti-IBDV antibodies, as detected by immunoblot and enzyme-linked immunosorbent assay tests. These antibodies did not prevent changes in the bursa and mortality when birds were challenged with a virulent IBDV strain after vaccination with the recombinant VP3. The results show that VP3 polypeptide cannot be used as a subunit vaccine against IBDV and raise questions concerning the nature of the neutralizing epitope on this structural protein.
Animals↗
[Cytomegalovirus diagnosis in immunocompromised patients: detection of early viral protein and viral nucleic acids in shell vial culture].
Conventional cell culture often requires several weeks for the detection of cytomegalovirus and is therefore an inappropriate technique for the rapid diagnosis of cytomegalovirus infections. Serology is not useful in the immunocompromised patient. We describe our experience with a rapid method for the detection of cytomegalovirus in known positive materials and in clinical specimens from immunocompromised patients. For this procedure the specimens were centrifuged onto cell monolayers. One day after infection early protein was detected by immunofluorescence, and in duplicate cultures viral DNA was detected by in situ hybridization after 5 days. Cytomegalovirus was found most often in urine and bronchoalveolar lavage, but less frequently (and to a lesser extent) in blood and saliva. No virus was found in stool and CSF. Cytomegalovirus was diagnosed only in 51.4% by the detection of early protein, whereas in situ hybridization detected cytomegalovirus in 93.1% of the specimens. For the diagnosis of cytomegalovirus we recommend the shell vial cell culture technique, followed by the detection of early proteins after 24 hours and in situ hybridization after 5 days.
Cytomegalovirus↗
Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription.
The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated amino-acylation of tRNA(Lys) is inhibited. Since tRNA(Lys) is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.
Acylation↗
Synthesis of vaccinia viral proteins in cytoplasmic extracts. II. Identification of early and late viral proteins.
The synthesis of vaccinia viral proteins has been studied in a cell-free system prepared from vaccinia virus-infected HeLa cells. The radioactively labeled proteins were identified as viral proteins by immunodiffusion, disc gel electrophoresis, and disc gel-immunoelectrophoresis. The cytoplasmic extracts, obtained from infected cells at different times during viral replication, synthesized the corresponding "early" or "late" viral proteins.
Autoradiography↗
Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus nonstructural protein 4 and viral protein (VP) 4, VP6, and VP7 in infants who received selected live oral rotavirus vaccines.
Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus enterotoxin nonstructural protein (NSP)-4, independent neutralization antigens viral protein (VP)-4 and VP7, and group A rotavirus common antigen VP6 were analyzed by an immunocytochemistry assay in infants who received 1 of several live oral rotavirus vaccines. Significant serum immunoglobulin (Ig) A and IgG antibody responses to homotypic and/or heterotypic NSP4s of genotype [A], [B], or [C] were detected after vaccination. The magnitude of antibody responses to homotypic and heterotypic NSP4s was not significantly different, irrespective of the NSP4 genotype of the administered vaccine strain. In addition, there were no significant differences between IgA antibody responses to homotypic and heterotypic VP7s. In contrast, IgA antibody responses to VP4 were predominantly homotypic. IgA antibody responses to VP7 were lower in magnitude than those to VP4 but were comparable to those to NSP4. Antibody titers to homotypic and/or heterotypic NSP4s were positively correlated with those to VP6 before and after vaccination.
Antibodies, Viral↗
Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.
Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR2 interaction may suggest a possible immunoregulatory role of HSV glycoprotein C.
Antibodies, Monoclonal↗
Antibodies against viral proteins can be produced effectively in response to the increased uptake of alpha 2-macroglobulin:viral protein conjugate by macrophages.
We have previously shown that foreign antigens conjugated to alpha 2-macroglobulin (alpha 2M) were effectively taken up by macrophages, which was followed by a remarkable increase in the proliferation of immune T cells. We present in this report that the production of antibodies against viral proteins can also be effectively achieved when viral proteins were conjugated to alpha 2M and fed to the in vitro immune system. Proteins derived from Kirstein murine sarcoma virus (Ki-MSV) were partially purified by gel chromatography and conjugated to alpha 2M by the action of proteinases. Viral proteins conjugated to alpha 2M were taken up by thioglycolate-induced murine peritoneal exudate cells (PEC) more effectively than the free viral proteins. Murine spleen cells were then added to PECs fed with free viral proteins or with alpha 2M: viral protein conjugates, and the cell mixtures were incubated for five days. Each culture medium was then assayed for a specific antibody production by enzyme-linked immunosorbent assay (ELISA). Figures based on such assays revealed that the production of antibodies against viral proteins was ten times higher when the proteins were fed to macrophages in conjugated forms with alpha 2M.
Animals↗
Identification of the vesicular stomatitis virus large protein as a unique viral protein.
Previous studies have noted the existence of a 190,000-dalton vesicular stomatitis virus (VSV) protein called the large (L) protein. To determine whether this protein is a nonspecific aggregate, a precursor to the other VSV proteins, or a unique viral protein, its synthesis relative to the other VSV proteins was studied under conditions of inhibition of initiation of protein synthesis. Also, its tryptic peptides were compared to those of the other VSV proteins. In both cases the results were consistent with the identification of the large protein as a unique viral protein.
Animals↗
The viral protein 3CD induces an equilibrium between the viral protein and RNA synthesis in a cell-free system for poliovirus replication.
In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log10. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log10 by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system.
3C Viral Proteases↗
A particulate viral protein vaccine reduces viral load and delays progression to disease in immunized ponies challenged with equine infectious anemia virus.
Immunization regimens that induce a broadly reactive cytolytic T lymphocyte (CTL) response specific for lentiviral antigens have emerged as the leading candidates in efficacy trials conducted in both animal modelshumans. To date, lentivirus vaccination strategies have overlooked one such immunization strategy, namely the use of particulate antigens. To evaluate the efficacy of targeting antigen into the phagocytic pathway to elicit a cell-mediated immune response to lentiviral antigens, we initiated the first study of a particulate-based vaccination protocol using a large animal model system. Gradient-purified equine infectious anemia virus (EIAV) was covalently coupled to glutaraldehyde-activated iron oxide beads. In vitro studies demonstrated the effectiveness of the inactivated whole virus particulate to prime antigen presenting cells for the activationexpansion of virus-specific CD8(+) CTL. The in vivo effectiveness of the particulate antigen was evaluated by experimental immunization of ponies. Ponies receiving the viral particulate vaccinechallenged with infectious EIAV had a delayed progression to diseasea reduced viral load compared with infected ponies that had not been vaccinated. Interestingly, in vitro virus-specific CTL activity was detected in only one of four immunized animals at the day of challenge. The beneficial effects of the particulate vaccine regimen were not clearly associated with any in vitro measurable parameters of the virus-specific cellular or humoral immune responses elicited by the vaccine at the day of challenge. However, within 3 weeks after virus challenge, anamnestic humoral responses characterized by a rapid emergence of neutralizing activity in the seruma predominance of conformationally dependent epitopes recognized by virus-specific antibodies were observed in the vaccinates. Taken together, further studies are clearly warranted in large animal model systems using a particulate-based vaccine regimen considering the beneficial effects of this regimen in our studythe protective effects of particulate antigen delivery in the murine model.
Animals↗
[Influenza virus proteins. Incorporation of newly synthesized viral proteins into virions].
Incorporation of individual viral proteins (P1, P2, P3, NP, NA, HA, and M) into virions was studied by the "pulse-chase" method followed by electrophoretic analysis of the proteins in chick fibroblast cultures infected with influenza WSN virus. Viral proteins were found to incorporate into virions rather rapidly and for a long time after synthesis, with the exception of M protein incorporation of which into the virus markedly decreased by 4 hours after synthesis. Nucleocapsid proteins (NP, P1, P2, and P3) incorporated into the virus asynchronously: by 17 hours postinoculation, in NP protein prevailed the molecules synthesized after 6 hours of infection, in P2 protein the molecules synthesized in the first hours of infection, and P1 and P3 proteins were represented by a uniform set of molecules synthesized at various periods after inoculation. The evidence on heterogeneity of P2 and M proteins in the virus has been obtained. Possible mechanisms regulating the selection of molecules of individual viral proteins in formation of virus particles are discussed.
Animals↗
Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.
Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.
Amino Acid Sequence↗
The in vitro of Rous sarcoma virus RNA and function of the viral protein during the viral replication.
The gag gene and pol gene of the Rous sarcoma virus are translated in vitro from the 35S viral RNA. The env gene cannot be translated in vitro from the 35S RNA. For the in vitro translation of the src gene. 3' end fragments of the viral RNA are used. The gag protein p15 has a proteolytic activity and specifically processes its own protein precursor pr76. The gag protein p19 suppresses the in vitro translation of the pol gene.
Animals↗