Use of electron microscopic and immunogold labeling techniques to determine polyomavirus recombinant VP1 capsid-like particles entry into mouse 3T6 cell nucleus.

Murine polyomavirus major structural protein VP1 could assemble into capsid-like particles when expressed in the baculovirus system. The recombinant capsid-like particles that were purified by CsCl density gradient centrifugation were capable of packaging host DNA. Electron microscopic and immunogold labeling techniques were used to study the entry of these VP1 recombinant capsid-like particles into mouse 3T6 cells. It was found that these VP1 recombinant capsid-like particles, which lack polyomavirus minor structural proteins (VP2 and VP3), use the same mechanism to enter mouse 3T6 cell cytoplasm and nucleus as that used by native polyomavirus virions.

Murine polyomavirus infection of 3T6 mouse cells shows evidence of predominant necrosis as well as limited apoptosis.

The current study was developed to determine if polyomavirus infected 3T6 mouse cells evoked an apoptotic or a necrotic mechanism during infection. Infected cells were analyzed by flow cytometry, transmission electron microscopy (TEM), DNA electrophoresis and by measuring caspase-3 enzymatic activity. Infected cells that were analyzed at 72 h post-infection showed the following: flow cytometry analysis revealed a 5% increase in apoptotic cells and a 46% increase in necrotic cells when compared to uninfected cells; electron microscopy showed 10% cells with characteristic apoptotic morphology and 40% with necrotic appearance; caspase-3 activity was found to increase two fold when compared to uninfected cells and DNA fragmentation (laddering) was clearly evident late in infection. It was concluded that infected cells predominantly showed necrosis, although some cells showed apoptosis in late infection. Recombinant capsid-like particles composed of the polyomavirus structural proteins were not able to induce cell death.

Use of the confocal microscope to determine polyomavirus recombinant capsid-like particle entry into mouse 3T6 cells.

The structural protein genes of polyomavirus were expressed in the baculovirus system, and the proteins were found to assemble into capsid-like particles capable of packaging insect cell DNA. Recombinant capsid-like particles could be produced that were composed of the various structural proteins (VP1, VP1/2, VP1/3 and VP1/2 + VP3). Laser scanning confocal microscopy was used to determine if the various capsid-like particles could infect (enter) mouse 3T6 cells. Each of the various capsid-like particles was equally capable of cell entry as determined by indirect immunofluorescence confocal microscopy.

Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid-like particles when expressed in the baculovirus system.

The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein-protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein-protein interaction between the expressed proteins. This protein-protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.

Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells.

Truncation of the nuclear localization signal of polyomavirus VP1 results in a loss of DNA packaging when expressed in the baculovirus system.

Using the pBlueBacIII baculovirus transfer vector, N11-VP1, a truncated form of the polyomavirus major capsid protein VP1, was cloned for expression in the baculovirus-insect cell expression system. The N11-VP1 protein is virtually identical to full-length, wild-type VP1, except that the first 11 amino acids have been deleted from the amino terminus of the protein. The N-terminal region of VP1 has previously been shown to contain the nuclear localization signal (NLS) of the protein and contains residues essential for both nuclear transport as well as DNA-binding functions. The 5-day infected Sf9 cellular lysate from the recombinant N11-VP1 preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as Western blotting and was shown to have accurately expressed the N11-VP1 as cloned. Examination of the Coomassie-stained gels revealed that the capsid-like particles composed of the N11-VP1 protein did not contain any host-derived histones. The absence of the histones in the N11-VP1 capsid-like particles is indicative of the inability of these particles to package DNA, a feature which is observed when wild-type VP1 is treated in this manner. Electron microscopy of these particles substantiated this observation. To determine if the deletion of the NLS exhibited true in vivo characteristics, Sf9 insect cells were infected with the recombinant baculovirus carrying the N11-VP1 gene and examined early in infection (30 h post-infection) by indirect immunofluorescence. The N11-VP1 protein was not transported to the nucleus and remained in the cytoplasm. When the Sf9 cells were coinfected with N11-VP1 and polyomavirus VP2 and VP3 carrying baculoviruses, the N11-VP1 was transported to the nucleus by cooperation with the minor capsid proteins. These studies demonstrate that the N-terminal region of VP1, which contains the NLS and DNA-binding domains, is essential for VP1 nuclear transport and its ability to package Sf9 cellular DNA.

Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line.

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Characterization of a calcium binding domain in the VP1 protein of the avian polyomavirus, budgerigar fledgling disease virus.

Calcium ions appear to play a major role in maintaining the structural integrity and assembly of papovavirus virions and are likely involved in the process of viral uncoating. Recently it was reported that the purified recombinant VP1 protein of budgerigar fledgling disease virus (BFDV) was capable of assembling into capsid-like particles in the presence of calcium. It is now reported that the major capsid protein VP1 of BFDV binds calcium ions in an in vitro calcium binding assay. Two deletions were made in the VP1 protein to identify a calcium binding domain and to further characterize the role of calcium ions in the capsid assembly process. Recombinant VP1 lacking a putative calcium binding domain (Asp-237-Asp-248) failed to bind radioactive 45Ca2+ yet associated into capsomeres. These capsomeres were similar in shape to the wild-type VP1 but were unable to assemble into capsid-like particles. Likewise, recombinant VP1 lacking ten carboxyl terminal amino acids (Glu-334-Arg-343) also formed capsomeres that were unable to assemble into capsid-like particles. In contrast to the VP1 protein with the internal deletion, the protein with the truncated carboxyl terminus bound 45Ca2+ in the in vitro assay. These results have identified a calcium binding domain (Asp-237-Asp-248) for the BFDV VP1 protein and a crucial role for the VP1 carboxyl terminal amino acids (Glu-334-Arg-343) in capsid assembly.

Methylation of the polyomavirus major capsid protein VP1.

Polyomavirus VP1 has been shown to be modified by phosphorylation, sulfation, acetylation and hydroxylation. The major capsid protein VP1 is now shown to be modified by methylation. Addition of cycloheximide to infected cultures followed by addition of [3H-methyl]-L-methionine and subsequent immunoprecipitation, SDS-PAGE and fluorography revealed methylation occurring on VP1. Amino acid analysis of [3H-methyl]-L-methionine-labelled polyomavirus VP1 by two-dimensional paper chromatography and HPLC of the acid-hydrolyzed protein revealed the presence of 3H-labelled trimethyllysine and monomethylarginine.

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Lipid and fatty acid analysis of uninfected and granulosis virus-infected Plodia interpunctella larvae.

A comparative study on the lipid and fatty acid composition of the uninfected and GV-infected Plodia interpunctella larvae was performed. Higher levels of free fatty acids were found in GV-infected larvae compared to those of the uninfected larvae, while the latter had more triacylglycerol compared to the former. The known identified phospholipids were fewer in the GV-infected larvae compared to those in the uninfected larvae. However, an unidentified phospholipid was found to be approximately two times higher in GV-infected larvae. The total lipid of both larvae had palmitic, oleic, and linoleic as the major fatty acids. The fatty acid composition of the GV-infected larval phospholipid differed considerably compared to that of the uninfected larvae, in that the ratio of unsaturated fatty acid to saturated fatty acid was 3.5 times less in the GV-infected larvae.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly.

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Infection with the avian polyomavirus, BFDV, selectively affects myofibril structure in embryonic chick ventricle cardiomyocytes.

Embryonic cardiomyocytes can both beat and divide. They assemble cardiac muscle-specific proteins into sarcomeric myofibrils and contract. In addition, they periodically synthesize DNA, complete mitosis, disassemble sarcomeric myofibrils in the area of the mitotic spindle, assemble cytoplasmic isoform-specific proteins into a cleavage furrow contractile ring, undergo cytokinesis, and then reform sarcomeric myofibrils in daughter cells. Little is known about how embryonic cardiomyocytes disassemble their myofibrils as they traverse the cell cycle and divide. In the present study, beating embryonic avian ventricular cardiomyocytes in primary culture were stimulated to initiate DNA synthesis without subsequent mitosis or cytokinesis by infection with the lytic avian polyomavirus, Budgerigar Fledgling Disease Virus (BFDV). Within 48 hours, infected, adherent cardiomyocytes disassemble most of their sarcomeric myofibrils, retaining cardiac myosin only in thin myofibrils with disrupted sarcomeric periodicity and in amorphous nonfibrillar pools. By 72 hours, infected cardiomyocytes contain no myofibrils and no longer react with antibodies to cardiac myosin. In contrast, infected cardiomyocytes continue to display cytoplasmic myosin localized in stress-fiber-like-structures in adherent cells, or in disrupted fibers and dispersed pools in detaching cells. Infected cardiomyocytes also continue to display interphase-like arrays of polymerized microtubules, even when rounded-up just prior to lysis. These results suggest that polyomavirus infection may provide a useful model system for further study of the regulation of myofibrils disassembly in embryonic cardiomyocytes.

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly.

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals.

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins.

Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus.

The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.

Virus protein assembly in microgravity.

The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid. VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure. Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly. Flight studies were conducted on STS-37 on April 5-9, 1991. Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0). After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation. Flight and ground-based samples were analyzed by electron microscopy. Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits. Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.