Levels of bovine papillomavirus RNA and protein expression correlate with variations in the tumorigenic phenotype of hamster cells

The methionine codon at bovine papillomavirus type 1 nucleotide 3091 was mutated to determine whether it may serve as an initiation codon for an E2 transcriptional repressor protein and to determine the role of the repressor in the biological activities of the virus. A series of transient expression experiments with CV1 cells documented that the mutation reduced expression of repressor activity from the viral genome and resulted in increased expression of the E5 transforming gene. Viral genomes containing the mutation displayed enhanced transforming activity in several assays in mouse C127 cells, including focus formation, colony formation in agarose, and tumorigenicity. In transformed cells, the mutant viral DNA was maintained as a plasmid with approximately 500 genomes per cell, whereas the wild-type copy number was approximately 75. These results indicate that the wild-type bovine papillomavirus type 1 genome encodes an E2 repressor protein that moderates the viral transforming activity and allows maintenance of the viral DNA at a relatively low copy number.

mentioning

confidence: 67%

Bovine papillomavirus E2 repressor mutant displays a high-copy-number phenotype and enhanced transforming activity

Riese

Settleman

Neary

et al. 1990

“…BPHE-1 cells were obtained from A. Lewis (National Institutes of Health, Bethesda, Md.) (42). C127 clone C cells were obtained from W. Vass (National Institutes of Health), and BHK-21 cells were from the American Type Culture Collection.…”

Section: Reagentsmentioning

confidence: 99%

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype

Roden

Greenstone

Kirnbauer

et al. 1996

265

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.

“…Subnuclear localization of BPV capsid proteins. BPHE-1 is a hamster fibroblast cell line that is latently infected with multiple copies of autonomously replicating BPV genomes and expresses the nonstructural viral proteins (57). We used the SFV expression system to introduce the L2 minor capsid protein into BPHE-1 cells and localized the L2 protein by immunofluorescence staining and laser scanning confocal microscopy.…”

Section: Resultsmentioning

confidence: 99%

“…Cell lines. BPHE-1 cells, obtained from Andrew Lewis Jr. (NIH), were grown in Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% fetal calf serum (57). BHK-21 cells were grown in Glascow's medium supplemented with 10% tryptose phosphate broth, antibiotics, nonessential amino acids, HEPES, and 5% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently reported a nonepithelial culture system for making infectious papillomavirus in which the contribution of certain viral genes to this process can be assessed (45). Infection of rodent fibroblasts with BPV virions leads to steady-state autonomous replication of the viral genome and transcription of only the nonstructural viral proteins, as is seen in the basal layers of the stratified squamous epithelium (35,48,57). Replication of the viral genome is dependent on two nonstructural proteins, E1 and E2, which specifically bind the viral DNA (1,8,42,54,56).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Papillomavirus Minor Capsid Protein, L2, Induces Localization of the Major Capsid Protein, L1, and the Viral Transcription/Replication Protein, E2, to PML Oncogenic Domains

Day

Roden

Lowy

et al. 1998

175

We have used immunofluorescent staining and confocal microscopy to examine the subcellular localization of structural and nonstructural bovine papillomavirus (BPV) proteins in cultured cells that produce infectious virions. When expressed separately, L1, the major capsid protein, showed a diffuse nuclear distribution while L2, the minor capsid protein, was found to localize to punctate nuclear regions identified as promonocytic leukemia protein (PML) oncogenic domains (PODs). Coexpression of L1 and L2 induced a relocation of L1 into the PODs, leading to the colocalization of L1 and L2. The effect of L2 expression on the distribution of the nonstructural viral proteins E1 and E2, which are required for maintenance of the genome and viral DNA synthesis, was also examined. The localization of the E1 protein was unaffected by L2 expression. However, the pattern of anti-E2 staining was dramatically altered in L2-expressing cells. Similar to L1, E2 was shifted from a dispersed nuclear locality into the PODs and colocalized with L2. The recruitment of full-length E2 by L2 occurred in the absence of other viral components. L2 was shown previously to be essential for the generation of infectious BPV. Our present results provide evidence for a role for L2 in the organization of virion components by recruiting them to a distinct nuclear domain. This L2-dependent colocalization probably serves as a mechanism to promote the assembly of papillomaviruses either by increasing the local concentration of virion constituents or by providing the physical architecture necessary for efficient packaging and assembly. The data also suggest a role for a nonstructural viral protein, E2, in virion assembly, specifically the recruitment of the viral genome to the sites of assembly, through its high-affinity interaction with specific sequences in the viral DNA.