Cottontail Rabbit Papillomavirus E8 Protein Is Essential for Wart Formation and Provides New Insights into Viral Pathogenesis

ABSTRACT The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.

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The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Journal of Virology ◽

10.1128/jvi.00384-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 5860-5875 ◽

Cited By ~ 21

Author(s):

Eva Maria Borst ◽

Rudolf Bauerfeind ◽

Anne Binz ◽

Thomas Min Stephan ◽

Sebastian Neuber ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Viral Genome ◽

Fluorescent Protein ◽

Unit Length ◽

Start Codon ◽

Nuclear Targeting ◽

Reading Frame ◽

A Genome ◽

Genome Encapsidation ◽

Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

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Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2615 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2615-2626 ◽

Cited By ~ 172

Author(s):

E Hickey ◽

S E Brandon ◽

G Smale ◽

D Lloyd ◽

L A Weber

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Normal Growth ◽

Gene Families ◽

Growth Conditions ◽

Complete Sequence ◽

Start Codon ◽

Hybridization Probe ◽

Reading Frame ◽

Alpha Gene

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

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cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/132.1.97 ◽

1992 ◽

Vol 132 (1) ◽

pp. 97-112 ◽

Cited By ~ 3

Author(s):

I Pinto ◽

J G Na ◽

F Sherman ◽

M Hampsey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Point Mutations ◽

Start Codon ◽

Reading Frame ◽

Codon Recognition ◽

Short Open Reading Frame ◽

Initiator Codon ◽

Extragenic Suppressors ◽

Cis And Trans

Abstract The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

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FuncPEP: A Database of Functional Peptides Encoded by Non-Coding RNAs

Non-Coding RNA ◽

10.3390/ncrna6040041 ◽

2020 ◽

Vol 6 (4) ◽

pp. 41

Author(s):

Mihnea P. Dragomir ◽

Ganiraju C. Manyam ◽

Leonie Florence Ott ◽

Léa Berland ◽

Erik Knutsen ◽

...

Keyword(s):

Open Reading Frames ◽

Start Codon ◽

Small Peptides ◽

Reading Frame ◽

Cellular Processes ◽

New Class ◽

Web Resource ◽

Non Coding Rnas ◽

Functional Peptides ◽

Small Open Reading Frames

Non-coding RNAs (ncRNAs) are essential players in many cellular processes, from normal development to oncogenic transformation. Initially, ncRNAs were defined as transcripts that lacked an open reading frame (ORF). However, multiple lines of evidence suggest that certain ncRNAs encode small peptides of less than 100 amino acids. The sequences encoding these peptides are known as small open reading frames (smORFs), many initiating with the traditional AUG start codon but terminating with atypical stop codons, suggesting a different biogenesis. The ncRNA-encoded peptides (ncPEPs) are gradually becoming appreciated as a new class of functional molecules that contribute to diverse cellular processes, and are deregulated in different diseases contributing to pathogenesis. As multiple publications have identified unique ncPEPs, we appreciated the need for assembling a new web resource that could gather information about these functional ncPEPs. We developed FuncPEP, a new database of functional ncRNA encoded peptides, containing all experimentally validated and functionally characterized ncPEPs. Currently, FuncPEP includes a comprehensive annotation of 112 functional ncPEPs and specific details regarding the ncRNA transcripts that encode these peptides. We believe that FuncPEP will serve as a platform for further deciphering the biologic significance and medical use of ncPEPs. The link for FuncPEP database can be found at the end of the Introduction Section.

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Lingrong Bai 1,†, Jing Wang 2,†, Huitong Zhou 3, Hua Gong 3, Jinzhong Tao 1,* and Jon G. H. Hickford 3,*

Animals ◽

10.3390/ani9040142 ◽

2019 ◽

Vol 9 (4) ◽

pp. 142 ◽

Cited By ~ 4

Author(s):

Lingrong Bai ◽

Jing Wang ◽

Huitong Zhou ◽

Hua Gong ◽

Jinzhong Tao ◽

...

Keyword(s):

Chromosome Region ◽

Mammalian Species ◽

Fibre Diameter ◽

Start Codon ◽

Wool Fibre ◽

Gene Marker ◽

Nucleotide Polymorphisms ◽

Repeat Polymorphism ◽

Reading Frame ◽

Associated Proteins

Keratin-associated proteins (KAPs) are a diverse group of proteins and form a matrix that cross-links keratin intermediate filaments in hair and wool fibres. From over 100 KAP genes (KRTAPs) identified in mammalian species, KRTAP25-1 is a high sulphur (HS)-KAP gene, which has recently been described in humans. Here, we report the absence of KRTAP25-1 in sheep, and describe a new HS-KRTAP (named KRTAP28-1) in the chromosome region where KRTAP25-1 was expected to be found. Six variants (A−F) of KRTAP28-1 containing eight single nucleotide polymorphisms (SNPs) and a TG repeat polymorphism were detected. One was positioned 30 bp upstream of the transcription start codon and all the others were non-synonymous SNPs, including a nonsense SNP. The TG repeat polymorphism would lead to a reading frame shift at the carboxyl-terminal end. The effect of KRTAP28-1 on wool traits was investigated with 383 Southdown × Merino-cross lambs from seven sire lines. Of the four genotypes with a frequency of over 5%, lambs of genotypes AB and BD produced wool of a smaller MFD than lambs of genotype BC. This shows that KRTAP28-1 is associated with wool fibre diameter, and that variation in this gene might have potential for use as a gene marker for reducing wool fibre diameter.

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Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits

Vaccine ◽

10.1016/s0264-410x(98)00356-9 ◽

1999 ◽

Vol 17 (11-12) ◽

pp. 1558-1566 ◽

Cited By ~ 29

Author(s):

R HAN

Keyword(s):

T Cell ◽

Immune Responses ◽

Plasmid Dna ◽

Intramuscular Injection ◽

Dna Encoding ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Cottontail Rabbit ◽

E6 And E7

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Regulation of the furA and catC Operon, Encoding a Ferric Uptake Regulator Homologue and Catalase-Peroxidase, Respectively, in Streptomyces coelicolor A3(2)

Journal of Bacteriology ◽

10.1128/jb.182.13.3767-3774.2000 ◽

2000 ◽

Vol 182 (13) ◽

pp. 3767-3774 ◽

Cited By ~ 37

Author(s):

Ji-Sook Hahn ◽

So-Young Oh ◽

Jung-Hye Roe

Keyword(s):

Streptomyces Coelicolor ◽

Transcriptional Start Site ◽

Protein Product ◽

Negative Regulator ◽

Start Codon ◽

Multicopy Plasmid ◽

Ferric Uptake Regulator ◽

Start Site ◽

Reading Frame ◽

Catalase Peroxidase

ABSTRACT We isolated the catC gene, encoding catalase-peroxidase in Streptomyces coelicolor, using sequence homology with the katG gene from Escherichia coli. Upstream of the catC gene, an open reading frame (furA) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the furA gene was cotranscribed with the catC gene. The transcriptional start site of the furA-catC mRNA was mapped to the translation start codon ATG of the furA gene. The putative promoter contains consensus −10 and −35 elements similar to those recognized by ςHrdB, the major sigma factor of S. coelicolor. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the furA gene was introduced into S. lividans on a multicopy plasmid, the increased production of catC transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the furA-catC operon. FurA protein bound to its own promoter region between −59 and −39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni2+, Mn2+, Zn2+, or Fe2+. Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.

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Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.

Blood ◽

10.1182/blood.v116.21.1444.1444 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1444-1444

Author(s):

Denisa Urban ◽

Ling Li ◽

James Wasmuth ◽

Hilary Christensen ◽

John Parkinson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Human Platelets ◽

Multiprotein Complex ◽

Yeast Two Hybrid ◽

Granule Formation ◽

Reading Frame ◽

Binding Partner ◽

Library Screen ◽

Two Hybrid ◽

Granule Release

Abstract Abstract 1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis. Disclosures: No relevant conflicts of interest to declare.

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