Biomedical subjects

Peter Tijssen

Publications and source records attributed to Peter Tijssen.

6 recordsLinked to original sources

PubMed2005

SAT: a late NS protein of porcine parvovirus.

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT(-)), with an unmodified VP sequence, showed a "slow-spreading" phenotype. These knockout mutants could be complemented with VP2(-) SAT(+) mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

Amino Acid Sequence↗

PubMed2004

Interfacial enzymology of parvovirus phospholipases A2.

The capsid of parvoviruses proteins were recently shown to contain secreted phospholipase A(2) (sPLA(2))-like activity that is required during host cell entry. Parvoviral PLA(2) domains have little sequence identity with sPLA(2)s and lack disulfide bonds. In the present study, after bacterial expression and purification, the biochemical characterizations of these first PLA(2)s identified in viruses have been investigated, and a comparison has been made with other known PLA(2)s. The specific activities of three viral PLA(2)s differed by 3 orders of magnitude, with porcine parvovirus PLA(2) displaying a specific activity similar to that of the most active sPLA(2)s (e.g. human group IIA) and the human AAV2 and B19 parvoviral enzymes displaying approximately 10(3) lower specific activities (similar to human sPLA(2) groups IIE and XIIA). These differences were not caused by weaker Ca(2+) or interfacial binding. The specific activities of the viral PLA(2)s on zwitterionic or anionic phospholipid vesicles were comparable. The viral PLA(2)s did not display a preference for unsaturated versus saturated sn-2 fatty acyl chains and hydrolyzed all major classes of glycero-phospholipids except phosphatidylinositol. Incubation of mammalian cells with porcine parvovirus PLA(2) led to the release of arachidonic acid into the culture medium. Interestingly, among nine previously known sPLA(2) inhibitors, only a subset showed inhibition of the viral PLA(2)s and with weak potency, indicating that the active sites of these new enzymes are structurally distinct from those of sPLA(2)s. Based on these distinct enzymatic and structural properties, we propose to classify the parvovirus PLA(2)s within the PLA(2) superfamily as group XIII enzymes.

Animals↗

PubMed2004

Construction and sequencing of an infectious clone of the human parvovirus B19.

Human parvovirus B19 has a nonenveloped, icosahedral capsid packaging a linear single-stranded DNA genome of 5.6 kb with long inverted terminal repeats (ITR) at both the 5' and 3' end. Previous attempts to construct a full-length B19 clone were unsuccessful due to deletions in the ITR sequences. We cloned the complete parvovirus B19 genome with intact ITRs from an aplastic crisis patient. Sequence analysis of the complete viral genome indicated that both 5' and 3' ITRs have two sequence configurations and several base changes within the ITRs compared to previous published sequences. After transfection of the plasmid into permissive cells, spliced and non-spliced viral transcripts and viral capsid proteins could be detected. Southern blot analysis of the DNA purified from the plasmid-transfected cells confirmed parvovirus B19 DNA replication. Production of infectious virus by the B19 plasmid was shown by inoculation of cell lysate derived from transfected cells into fresh cells. Together, these results indicate the first successful production of an infectious clone for parvovirus B19 virus.

3' Untranslated Regions↗

PubMed2004

A parvovirus isolated from royal python (Python regius) is a member of the genus Dependovirus.

Parvoviruses were isolated from Python regius and Boa constrictor snakes and propagated in viper heart (VH-2) and iguana heart (IgH-2) cells. The full-length genome of a snake parvovirus was cloned and both strands were sequenced. The organization of the 4432-nt-long genome was found to be typical of parvoviruses. This genome was flanked by inverted terminal repeats (ITRs) of 154 nt, containing 122 nt terminal hairpins and contained two large open reading frames, encoding the non-structural and structural proteins. Genes of this new parvovirus were most similar to those from waterfowl parvoviruses and from adeno-associated viruses (AAVs), albeit to a relatively low degree and with some organizational differences. The structure of its ITRs also closely resembled those of AAVs. Based on these data, we propose to classify this virus, the first serpentine parvovirus to be identified, as serpentine adeno-associated virus (SAAV) in the genus Dependovirus.

Amino Acid Sequence↗

PubMed2002

The structure of porcine parvovirus: comparison with related viruses.

The structure of baculovirus-expressed porcine parvovirus (PPV) capsids was solved using X-ray crystallography and was found to be similar to the related canine parvovirus (CPV) and minute virus of mice (MVM). The PPV capsid protein has 57 % and 49 % amino acid sequence identity with CPV and MVM, respectively, but the degree of conservation of surface-exposed residues is lower than average. Consequently, most of the structural differences are on the surface and are the probable cause of the known variability in antigenicity and host range. The NADL-2 and Kresse strains of PPV have distinct tissue tropisms and pathogenicity, which are mediated by one or more of the amino acid residues 381, 386, and 436. These residues are on or near the surface of the virus capsid, where they are likely to be associated with virus-cell interactions.

Amino Acid Sequence↗

PubMed2002

The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity.

The unique region of the VP1 protein of parvoviruses was proposed to contain a parvoviral phospholipase A2 (pvPLA2) motif. Here, PLA2 activity is shown in the unique region of adeno-associated virus type 2 (AAV-2) VP1 when expressed as an isolated domain in bacteria. Mutations in this region of the capsid protein strongly reduced the infectivity of mutant virions in comparison to wild-type AAV-2. This correlated with effects on the activity of PLA2. The mutations had no influence on capsid assembly, packaging of viral genomes into particles or binding to and entry into HeLa cells. However, a delayed onset and reduced amount of early gene expression, as measured by Rep immunofluorescence, was observed. These results suggest that pvPLA2 activity is required for a step following perinuclear accumulation of virions but prior to early gene expression.

Capsid↗