Human Polyomavirus Infection in Renal Allograft Recipients

Cytological and virological studies on 74 patients with functioning renal allografts were undertaken to detect polyomavirus infection of the renal tract. Ten patients (13·5%) were excreting polyomavirus. Virus particles were seen in the electron microscope in urine samples from eight patients. B.K. polyomavirus was isolated from four patients. Infection with a different polyomavirus was probable in one patient. Virus isolation was most readily achieved when large numbers of intact virus particles were seen in the urine. Patients who were excreting large amounts of polyomavirus shed numerous inclusion-bearing cells which could be detected by cytology. A serological study showed that the prevalence of B.K. antibody was similar to that found in the general population and 38% of this series of transplant patients had evidence of active infection with B.K. virus.     

WU多瘤病毒在中国浙江地区的发现与鉴定

呼吸道病毒种类很多,大部分的病毒感染机体后可以导致大面积人群不同程度的发病,严重的可导致死亡[1].     

Translational efficiencies of polyomavirus late mRNA molecules that differ in the sequences of their 5'' noncoding late leader exons.

Mouse NIH 3T6 cells were coinfected with two strains of polyomavirus that differ only in the sequences of their 5' noncoding late leader exons. Polysomes were isolated at late times after infection and probed with oligonucleotides specific for each strain. Results indicate that the sequence of the late leader does not play a role in the translational efficiency of late polyomavirus messages.     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

DNAs of two molecularly cloned endogenous ecotropic proviruses are poorly infectious in DNA transfection assays

We have isolated a series of point mutants in the polyomavirus origin-intergenic control region by using a procedure which exploits the single-stranded nature of DNAs cloned into M13 phage both for the generation of mutants and for their analysis by DNA sequencing. In this report we describe the effects of cytosine X guanine----thymine X adenine base-pair substitutions in the polyomavirus origin region upon replication in mouse 3T6 cells of the M13-polyomavirus constructs. Our results indicate that sequences near the center of a 34-base-pair sequence with dyad symmetry are important for replication, whereas specific nucleotides near the ends of the dyad symmetry are not important. Furthermore, a putative large T antigen-binding site at nucleotides 60 to 80 can be mutated without affecting replication function as measured in this assay.     

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.     

Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.     

A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line.     

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.     

Multiple domains in the polyomavirus B enhancer are required for productive infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.     

Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences.

Polyomavirus mutants were isolated from PCC4 embryonal carcinoma cells infected with a variant strain of polyomavirus (ev 1001h) and were found to contain a tandem duplication overlapping the enhancers and the origin of replication. These mutants were able to infect several lines of embryonal carcinoma cells, including PCC4, F9, and LT1. The sequence and structure of one of these mutants are presented and compared with those of other PyEC PCC4 mutants previously described.     

Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor.

We used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.