Targeting the retinoblastoma protein by MC007L, gene product of the molluscum contagiosum virus: detection of a novel virus-cell interaction by a member of the poxviruses.

The human pathogenic poxvirus molluscum contagiosum virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxvirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a virus can perturb the cell cycle.

Envelope targeting: hemagglutinin attachment specificity rather than fusion protein cleavage-activation restricts Tupaia paramyxovirus tropism.

To engineer a targeting envelope for gene and oncolytic vector delivery, we characterized and modified the envelope proteins of Tupaia paramyxovirus (TPMV), a relative of the morbilli- and henipaviruses that neither infects humans nor has cross-reactive relatives that infect humans. We completed the TPMV genomic sequence and noted that the predicted fusion (F) protein cleavage-activation site is not preceded by a canonical furin cleavage sequence. Coexpression of the TPMV F and hemagglutinin (H) proteins induced fusion of Tupaia baby fibroblasts but not of human cells, a finding consistent with the restricted TPMV host range. To identify the factors restricting fusion of non-Tupaia cells, we initially analyzed F protein cleavage. Even without an oligo- or monobasic protease cleavage sequence, TPMV F was cleaved in F1 and F2 subunits in human cells. Edman degradation of the F1 subunit yielded the sequence IFWGAIIA, placing the conserved phenylalanine in position 2, a novelty for paramyxoviruses but not the cause of fusion restriction. We then verified whether the lack of a TPMV H receptor limits fusion. Toward this end, we displayed a single-chain antibody (scFv) specific for the designated receptor human carcinoembryonic antigen on the TPMV H ectodomain. The H-scFv hybrid protein coexpressed with TPMV F mediated fusion of cells expressing the designated receptor, proving that the lack of a receptor limits fusion and that TPMV H can be retargeted. Targeting competence and the absence of antibodies in humans define the TPMV envelope as a module to be adapted for ferrying ribonucleocapsids of oncolytic viruses and gene delivery vectors.

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

DNA polymerase gene locus of Cercopithecine herpesvirus 1 is a suitable target for specific and rapid identification of viral infection by PCR technology.

The family Herpesviridae comprises at least 100 herpesviruses. Numerous human and animal pathogenic herpesviruses have been identified so far, including Cercopithecine herpesvirus 1 (CeHV-1). This virus is a member of the subfamily Alphaherpesvirinae and is the most hazardous herpesvirus to man. CeHV-1 is also known as B-virus or monkey B virus and as Herpesvirus simiae. In order to gain more genetic information, the viral DNA polymerase (DPOL) gene was identified using polymerase chain reaction (PCR) and DNA nucleotide sequence analysis. The deduced amino acid sequence contains the motifs and signatures that are typical for the B-family of DPOLs. The DPOL gene of CeHV-1 was found to be a suitable target for the specific and rapid identification of the Cercopithecine herpesvirus 1 infection by PCR technology. Comparative analysis of the DNA sequences of the DPOL gene loci of CeHV-1, Human herpesvirus 1 and 2 (HHV-1 and HHV-2), and other herpesviruses was carried out for determination of unique genomic regions of the individual DPOL genes. A primer set of 12 primers was used for screening the DNA of CeHV-1, HHV-1, and HHV-2 by detailed PCR. It was found that six out of twelve primer combinations are able to detect specifically the CeHV-1 genome without cross reactivity with the genome of HHV-1 and/or HHV-2. The specificity of the individual amplified DNA fragments was confirmed by DNA nucleotide sequence analysis. The results of these studies indicate that the six primer combinations of the specific CeHV-1 DPOL primer set is the method of choice for a rapid, precise and specific identification of a CeHV-1 infection by PCR. Due to the fact that this specific CeHV-1 DPOL primer set does not amplify any DNAs of HHV-1 or HHV-2 genome this technology is stressing and can be successfully used unlimited and more credible in all laboratories with PCR technical facility routinely for detection of a CeHV-1 infection in vivo or in vitro.

New ecological aspects of hantavirus infection: a change of a paradigm and a challenge of prevention--a review.

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.

Testing the possibility to protect bovine PrPC transgenic Swiss mice against bovine PrPSc infection by DNA vaccination using recombinant plasmid vectors harboring and expressing the complete or partial cDNA sequences of bovine PrPC.

The objective of this study was to investigate the molecular mechanisms of neurobiological processes involved in the degeneration of the central nervous system. The bovine spongiform encephalopathy (BSE) was used as experimental model system for investigation of transmissible spongiform encephalopathy (TSE). The experimental strategy was to evaluate the possibility for protection of bovine PrP(C) transgenic mice against a bovine PrP(Sc) infection by DNA vaccination using the complete or partial cDNA sequences of the bovine prion protein. Three recombinant plasmids pCR3.1-EX-PrP-BSE-C20 (C20), pCR3.1-EX-PrP-BSE-90-235-C4 (C4), and pCR3.1-EX-PrP-BSE-106-131-C14 (C14) were constructed. These mammalian expression vectors harbor complete (C20) or partial (C4 and C14) cDNA sequences of the Bos taurus PrP(C) (BTPrP(C)) encoding for amino acid residues 1-264 (C20), 90-235 (C4), and 106-131 (C14) of the BTPrP(C). Transgenic mice harboring and expressing BTPrP(C) were generated using the donor strain C57/CBA, receptor strain Swiss mouse, and recombinant plasmid MoPrPXho-boPrP. Crossing of positive transgenic mice to bovine PrP and negative to murine PrP with 129/OLA (murine PrP-/-) and C57BL6x129/OLA (murine PrP+/-) mice was carried out to amplify the colony of transgenic mice termed bovine PrP(C) transgenic Swiss mice (BTPrP-TgM). The capabilities of C20, C4, and C14 to express the corresponding cDNA sequence of BTPrP(C) in vitro and in vivo were confirmed prior to DNA vaccination of the BTPrP-TgM using NIH 3T3 cells and BALB/c mice, respectively. In order to prove the capability of the constructed expression vectors to protect BTPrP-TgM in vivo against a BSE infection 80 female BTPrP-TgM were vaccinated intramuscularly and subcutaneously with DNA of the plasmids C20, C4, C14, and parental vector pCR3.1 (100 microg DNA corresponding to about 26-30 pmol DNA/animal and application) in four groups (each consists of 20 animals). DNA vaccination was followed by three additional boosters. The vaccinated animals (15 animals of each group) were challenged twice per oral with homogenates of brain material obtained from BSE cattle containing the infectious PrP(Sc) (100 microl/animal which corresponds to 15 mg of a 15% brain homogenate). The first and second challenge experiments were performed 76-83 and 181 days post DNA vaccination, respectively. A part of the vaccinated animals (3-5 animals of each group) that served as internal negative control were mock infected using the brain homogenate of healthy cattle or Phosphate saline buffer (PBS). A variety of symptoms and clinical pictures were observed during the monitoring of DNA vaccinated animals. However, the observed diseases seem to be similar in all experimental animal groups. After an observation period of 14 months post the second challenge experiment the remaining animals (some animals died or were sacrificed when moribund during the study) were sacrificed after expiration of the experimental schedule. The right hemisphere of the brain and a half of the spleen tissue of the individual animals were used for detection of PrP(Sc) by Western blot analysis. The misfolded bovine PrP(Sc) was not detected in the brain or spleen tissues of those animals that were vaccinated with DNA of C20, which was able to express the complete bovine PrP(C) protein in vitro and in vivo. In contrast, the bovine PrP(Sc) was detected in the brain or spleen tissues of animals that were DNA vaccinated with DNA of the parental vector pCR3.1, with DNA of C4, or with DNA of C14. The results of these studies underline that the constructed expression vector C20 possesses the protective capacity to inhibit the formation of misfolded bovine PrP(Sc) in BTPrP-TgM under the conditions used. A delay of occurrence of TSE-specific symptoms in the majority of the vaccinated animals seems to be due to the prolonged incubation time of BSE infection.

Puumala virus nucleocapsid protein expressed in transgenic plants is not immunogenic after oral administration.

Transgenic plants expressing foreign genes are suitable systems for the production of relevant immunogens in high amounts that can be used to develop a new generation of vaccines against a variety of infectious diseases. Transgenic tobacco and potato plants expressing the nucleocapsid protein of Hantavirus serotype Puumala were generated and established. Puumala virus is a human pathogen causing hemorrhagic fever with renal syndrome. To investigate oral immunogenicity of the nucleocapsid protein expressed in plants, mice were fed with tubers of transgenic potato and tobacco leaf powder. The resulting antibodies were compared among groups. No significant difference could be found between the control group and the groups of animals, which had been fed with the recombinant plants expressing Puumala nucleocapsid protein. Hence, the effect of different enzymes, present in the gastro-intestinal tract, on the plant-derived antigen was investigated. It was found that the recombinant viral protein was completely degraded by trypsin and/or pepsin. In conclusion, the enzymes present in the intestine can degrade major antigenic domains of antigens, expressed in transgenic plants, thus preventing the induction of antibodies against the ingested viral antigen.

Re-evaluation and in silico annotation of the Tupaia herpesvirus proteins.

Herpesviruses represent an exceptionally suitable model to analyze evolutionary old pathogens, their competency to adapt to existing and changing molecular niches in host species, and the modulation of the gene content and function to comply with the requirements of life. The basis for numerous studies dealing with these questions are reliable statements about the gene content of herpesviral genomes and the functions of viral proteins. The recent determination of the coding strategy of the chimpanzee cytomegalovirus genome and the re-evaluation of the gene content of the human cytomegalovirus genome made it also necessary to restructure the putative transcription map of the Tupaia herpesvirus (THV) genome. Twenty-three THV-specific ORFs formerly predicted to be coding for viral proteins were deleted from the THV transcription map resulting in a gene layout that is now characterized by the presence of conserved genes in the genome center, that probably reflect the genome structure of common herpesviral ancestors, and species-specific genes at the termini. The conserved regions in the THV genome are characterized by high G + C contents between 60% and 80%, a high CpG dinucleotide frequency, and the presence of densely packed putative CpG islands. The genome termini seem to provide the requirements of large scale rearrangements and complements of the gene content to adapt to new environmental demands. With the help of the recently designed method of dictionary-driven, pattern-based protein annotation it was possible to assign putative functions to almost all potential THV proteins, e.g. 123 were found to be putative membrane or secreted proteins, putative signal domains were identified in 69, and 29 proteins were predicted to be glycosylated. The present study adds new aspects to the knowledge about the precise gene composition of herpesvirus genomes and viral protein functions that are of exceptional importance for studies dealing with the phylogeny, the evolution, vaccine vector development, virus-host interactions, pathogenesis and the determination of protein functions of herpesviruses.

Molecular anatomy of Tupaia (tree shrew) adenovirus genome; evolution of viral genes and viral phylogeny.

Adenoviruses are globally spread and infect species in all five taxons of vertebrates. Outstanding attention is focused on adenoviruses because of their transformation potential, their possible usability as vectors in gene therapy and their applicability in studies dealing with, e.g. cell cycle control, DNA replication, transcription, splicing, virus-host interactions, apoptosis, and viral evolution. The accumulation of genetic data provides the basis for the increase of our knowledge about adenoviruses. The Tupaia adenovirus (TAV) infects members of the genus Tupaiidae that are frequently used as laboratory animals in behavior research dealing with questions about biological and molecular processes of stress in mammals, in neurobiological and physiological studies, and as model organisms for human hepatitis B and C virus infections. In the present study the TAV genome underwent an extensive analysis including determination of codon usage, CG depletion, gene content, gene arrangement, potential splice sites, and phylogeny. The TAV genome has a length of 33,501 bp with a G+C content of 49.96%. The genome termini show a strong CG depletion that could be due to methylation of these genome regions during the viral replication cycle. The analysis of the coding capacity of the complete TAV genome resulted in the identification of 109 open reading frames (ORFs), of which 38 were predicted to be real viral genes. TAV was classified within the genus Mastadenovirus characterized by typical gene content, arrangement, and homology values of 29 conserved ORFs. Phylogenetic trees show that TAV is part of a separate evolutionary lineage and no mastadenovirus species can be considered as the most related. In contrast to other mastadenoviruses a direct ancestor of TAV captured a DUT gene from its mammalian host, presumably controlling local dUTP levels during replication and enhance viral replication in non-dividing host tissues. Furthermore, TAV possesses a second DNA-binding protein gene, that is likely to play a role in the determination of the host range. In view of these data it is conceivable that TAV underwent evolutionary adaptations to its biological environment resulting in the formation of special genomic components that provided TAV with the ability to expand its host range during viral evolution.

Characterization of the complete genome of the Tupaia (tree shrew) adenovirus.

The members of the family Adenoviridae are widely spread among vertebrate host species and normally cause acute but innocuous infections. Special attention is focused on adenoviruses because of their ability to transform host cells, their possible application in vector technology, and their phylogeny. The primary structure of the genome of Tupaia adenovirus (TAV), which infects Tupaia spp. (tree shrew) was determined. Tree shrews are taxonomically assumed to be at the base of the phylogenetic tree of mammals and are frequently used as laboratory animals in neurological and behavior research. The TAV genome is 33,501 bp in length with a G+C content of 49.96% and has 166-bp inverted terminal repeats. Analysis of the complete nucleotide sequence resulted in the identification of 109 open reading frames (ORFs) with a coding capacity of at least 40 amino acid residues. Thirty-eight of them are predicted to encode viral proteins based on the presence of transcription and translation signals and sequence and positional conservation. Thirty viral ORFs were found to show significant similarities to known adenoviral genes, arranged into discrete early and late genome regions as they are known from mastadenoviruses. Analysis of the nucleotide content of the TAV genome revealed a significant CG dinucleotide depletion at the genome ends that suggests methylation of these genomic regions during the viral life cycle. Phylogenetic analysis of the viral gene products, including penton and hexon proteins, viral protease, terminal protein, protein VIII, DNA polymerase, protein IVa2, and 100,000-molecular-weight protein, revealed that the evolutionary lineage of TAV forms a separate branch within the phylogenetic tree of the Mastadenovirus genus.

Characterization of expression of Puumala virus nucleocapsid protein in transgenic plants.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 microg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest.

Molecular anatomy of Chilo iridescent virus genome and the evolution of viral genes.

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482 bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.