- Volume 80, Issue 4, 1999

Three early proteins expressed by adenovirus type 5, E1b 55K, E4 Orf3 and E4 Orf6, are involved in regulating late viral gene expression. It has previously been shown that 55K associates with Orf6. Here we show that 55K also associates with Orf3 and that this interaction is necessary for 55K to localize to the nuclear matrix fraction of the cell. From our data, we infer that the Orf3 and Orf6 interactions with 55K may be mutually exclusive. The Orf3 protein is also known to associate with and cause the reorganization of cell nucleus structures known as ND10 or PODs. Consistent with the observed increase in the biochemical interaction between 55K and Orf3 in the absence of Orf6, the 55K association with Orf3 in ND10 was also found to increase in the absence of Orf6. The most studied cellular component of ND10 is PML, a complex protein present in a range of isoforms, some of which are modified by conjugation to the small ubiquitin-like protein PIC-1. The pattern of PML isoforms was altered in adenovirus-infected cells, in that a number of additional isoform bands appeared in an Orf3-dependent manner, one of which became predominant later in infection. As for ND10 reorganization, neither Orf6 nor 55K was required for this effect. Therefore it is likely that these changes in PML are related to the changes in ND10 structure that occur during infection.

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.

Two forms of human polyomavirus JC (JCV) genome are known based upon the structure of the transcriptional control region (TCR) of the virus: the archetypal form, which is commonly detected in urine, and the rearranged form, which was first detected in brain tissue from progressive multifocal leukoencephalopathy (PML) patients. The latter actually includes a group of TCR variants that, relative to the former, are characterized by various deletions and/or duplications. The aim of this study was to establish whether or not a correlation exists among the TCR type, the spreading of the virus within the host and its ability to cause PML. JCV TCR sequences from peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) obtained from various groups of patients were compared. JCV with archetypal TCR was detected in CSF and PBL specimens from patients without neurological disorders or who eventually received a diagnosis of a non-PML neurological disorder. Rearranged TCR sequences were detected in all the CSF and PBL specimens from PML patients. The high similarity observed between the TCR structure detected in PBL and CSF specimens from individual patients could strengthen the hypothesis that PBL has a role in spreading JCV to the brain. Moreover, heterogeneous TCR patterns have been shown in individual PBL specimens from PML patients. This supports the hypothesis that, in PBL, JCV may replicate and undergo rearrangements of the TCR. The detection of JCV DNA by PCR in CSF independently from PML, although rare, could suggest that this assay is not sufficient for a virological diagnosis of PML. Further studies are required to assess the usefulness of quantitative assays or TCR typing in combination with PCR for diagnostic purposes.

Human papillomavirus type 6 (HPV-6) is the causative agent of condyloma acuminata, a common sexually transmitted disease. Virus-like particles (VLPs) assembled from the L1 major capsid protein represent promising candidates for prophylactic vaccines. However, any intratype sequence variation among HPV-6 L1 ORFs will influence which sequence is used for a vaccine according to its prevalence in the population and its propensity for VLP production. Therefore, we have analysed the entire L1 nucleotide sequence of 17 clinical isolates of HPV-6 from the London area. We found 28 positions where changes from the prototype HPV-6b L1 occurred, showing that HPV-6 L1 intratype variation is greater than previously reported. The most frequently observed substitutions are clustered into three discrete regions: R1 (nt 5920-6075), R2 (nt 6590-6670) and R3 (nt 7070-7230). Indeed, most of the nucleotide substitutions within the HPV-6 L1 reported worldwide also map to these regions. The R3 region contains predominantly non-silent substitutions, the most common of which is a G-to-C substitution at position 7079. This results in a Glu-to-Gln change at aa 431, although this change had no effect on VLP yield or stability. This substitution defines a new HPV-6 L1 amino acid sequence that is more abundant in the isolates examined than any other reported sequence.

The long control region (LCR) and the E2 protein of human papillomaviruses (HPV) are the most important viral factors regulating transcription of the viral oncogenes E6 and E7. Sequence variation within these genomic regions may have an impact on the oncogenic potential of the virus. Sequence variation in the LCR and in the E2 gene of human papillomavirus type 16 (HPV-16) isolates originating from cervical cancer patients from East Hungary was studied. In 30 samples, sequencing and/or single-strand conformation polymorphism analysis revealed variants belonging to the European variant lineage of HPV-16. These variants differed from the reference European clone only slightly in their E2 and LCR sequences. Three samples represented variants belonging to the Asian-American group. These differed from the published reference sequence at several positions in the LCR and E2 regions. Compared to the reference clone, the LCR clones of the European isolates showed very similar transcriptional activities, while that of an Asian-American isolate had ~ 1.7-fold increased activity. Most of the increased activity of the Asian-American isolate could be ascribed to nucleotide changes found at the 3′ end of the LCR (nt 7660-7890). The transcriptional transactivation potentials of the HPV-16 E2 isolates differed only slightly from each other, and the differences seemed to be independent of the taxonomic position of the isolates.

The role of polyhedrin in the occlusion of virions was studied by substituting two heterologous polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.

We have examined the host range in different insect cell lines of Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants lacking p35, iap1 or iap2. These genes encode, or are predicted to encode, anti-apoptotic proteins. Abrogation of p35 reduced the ability of AcMNPV to replicate in permissive cell lines derived from Spodoptera frugiperda insects by inducing apoptosis. In semi-permissive cell lines, such as Lymantria dispar and Spodoptera littoralis cells, we observed cytopathic effects after infection with AcMNPV but little virus production. Infection of these cells by AcMNPV lacking p35 resulted in apoptosis. However, p35-deficient viruses were still able to replicate normally in Trichoplusia ni, Mamestra brassicae and Panolis flammea cell lines. Disruption of AcMNPV iap1 and iap2 was found not to affect virus replication in any of the cell lines. It was also possible to disrupt both iap1 and iap2 in the same virus without loss of infectivity. A virus without iap1 and p35 demonstrated identical growth characteristics and host range to a virus lacking p35. We conclude that in cells which respond to AcMNPV infection by initiating programmed cell death, the p35 gene product alone is sufficient to inhibit apoptosis. Removal of iap1 or iap2 has no effect on virus replication, even in cell lines which do not undergo apoptosis in response to AcMNPV infection. Our results with two semi-permissive cell lines further indicate that whilst p35 is important in blocking block apoptosis, other factors are involved in restricting AcMNPV replication within these cells.

We have started to characterize the capsid components of European Chlorella virus isolate CVG-1, a member of the Pbi subgroup of the Phycodnaviridae. The major coat protein, Vp49, was biochemically characterized and the amino acid sequence of the N terminus was determined. Subsequently, the corresponding gene was isolated from CVG-1 genomic DNA. Sequence data were compared to those available from PBCV-1 and other Chlorella virus isolates representing the NC64A subgroup of the Phycodnaviridae. The major coat proteins of all strains are homologous and similar in size, but apparently differ in their degree of glycosylation. Like PBCV-1, the major coat protein of CVG-1 is part of a gene family, as two open reading frames with high similarity to Vp49 were also isolated and characterized in this study. The predicted amino acid sequences of the CVG-1 and PBCV-1 virus genes examined show, with one exception, a divergence of about 25%. Taking into account that corresponding genes of NC64A viruses are almost identical, this divergence supports the original placement of the NC64A and Pbi viruses into separate subgroups of the Phycodnaviridae.

Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.

Amphotericin B (AmB) has been shown to delay hamster scrapie. Infectivity studies have been performed previously using AmB in order to understand the relationship between the accumulation of an abnormal isoform (PrPres) of the prion protein and 263K scrapie agent replication in the brain. The first study reported that AmB had no effect upon agent replication, although it delayed the development of both clinical signs and PrPres accumulation. However, subsequent experiments using the same model showed a significant effect both on agent replication and PrPres accumulation early in infection. This fundamental discrepancy was assumed to be linked to differences in experimental protocols. In order to unravel the issue, a new experiment has been performed encompassing different protocols and using an AmB derivative, MS-8209, that can be used at higher doses because of its lower toxicity. The findings of this study exclude the suspected differences in the protocols as the reason for previous conflicting results, and suggest strongly that these discrepancies were due to a low dose of AmB causing a ‘threshold effect’. Overall, this study indicates that, in this model, PrPres cannot be dissociated from infectivity by polyene antibiotics.