Zoltán Zádori

A Viral Phospholipase A2 Is Required for Parvovirus Infectivity

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.

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.

Organization and Expression Strategy of the Ambisense Genome of Densonucleosis Virus of Galleria mellonella

ABSTRACT The expression strategy of parvoviruses of the Densovirus genus has as yet not been reported. Clones were obtained from the densonucleosis virus of Galleria mellonella (GmDNV) that yielded infectious virus upon transfection into LD652 cells. Its genome was found to be the longest (6,039 nucleotides [nt]), with the largest inverted terminal repeats (ITRs) (550 nt) among all parvoviruses. The distal 136 nt could be folded into hairpins with flop or flip sequence orientations. In contrast to vertebrate parvoviruses, the gene cassettes for the nonstructural (NS) and structural (VP) proteins were found on the 5′ halves of the opposite strands. The transcripts for both cassettes started 23 nt downstream of the ITRs. The TATA boxes, as well as all upstream promoter elements, were localized in the ITRs and, therefore, identical for the NS and VP transcripts. These transcripts overlapped for 60 nt at the 3′ ends (antisense RNAs) at 50 m.u. The NS cassette consisted of three genes of which NS2 was contained completely within NS1 but from a different reading frame. Most of the NS transcripts were spliced to remove the upstream NS3, allowing leaky scanning translation of NS1 and NS2, similar to the genes of RNA-6 of influenza B virus. NS3 could be translated from the unspliced transcript. The VP transcript was not spliced and generated four VPs by a leaky scanning mechanism. The 5′-untranslated region of the VP transcript was only 5 nt long. Despite the transcription and translation strategies being radically different from those of vertebrate parvoviruses, the capsid was found to have phospholipase A2 activity, a feature thus far unique for parvoviruses.

Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid.

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca(2+)-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

Characteristics of the genome of goose parvovirus

The nucleic acid of goose parvovirus showed sensitivity to DNase and Mung Bean nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double-stranded form of about 5000 base pairs. There was evidence for encapsidation of strands of opposite polarities in equal amounts. The restriction enzyme cleavage patterns of goose and muscovy duck parvovirus DNAs differed significantly. However, they showed a high degree of homology by a hybridization test.

SAT: a Late NS Protein of Porcine Parvovirus

ABSTRACT 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.

The Acheta domesticus Densovirus, Isolated from the European House Cricket, Has Evolved an Expression Strategy Unique among Parvoviruses

ABSTRACT The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses.

The Expression Strategy of Goose Parvovirus Exhibits Features of both the Dependovirus and Parvovirus Genera

ABSTRACT The RNA transcription profile of the goose parvovirus (GPV) was determined, and it is a surprising hybrid of features of the Parvovirus and Dependovirus genera of the Parvovirinae subfamily of the Parvoviridae. Similar to the Dependovirus adeno-associated virus type 5, RNAs transcribed from the GPV upstream P9 promoter, which encode the viral nonstructural proteins, were polyadenylated at a high efficiency at a polyadenylation site [(pA)p] located within an intron in the center of the genome. Efficient usage of (pA)p required a downstream element that overlaps with the polypyrimidine tract of the A2 3′ splice site of the central intron. An upstream element required for efficient use of (pA)p was also identified. RNAs transcribed from the P42 promoter, presumed to encode the viral capsid proteins, primarily extended through (pA)p and were polyadenylated at a site, (pA)d, located at the right end of the genome and ultimately spliced at a high efficiency. No promoter analogous to the Dependovirus P19 promoter was detected; however, similar to minute virus of mice and other members of the Parvovirus genus, a significant portion of pre-mRNAs generated from the P9 promoter were additionally spliced within the putative GPV Rep1 coding region and likely encode an additional, smaller, nonstructural protein. Also similar to members of the Parvovirus genus, detectable activity of the GPV P42 promoter was highly dependent on transactivation by the GPV Rep1 protein in a manner dependent on binding to a cis-element located in the P42 promoter.

Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism

ABSTRACT The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log10 titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.

Four New Inverted Terminal Repeat Sequences from Bovine Adenoviruses Reveal Striking Differences in the Length and Content of the ITRs

The inverted terminal repeat (ITR) of the genome of four bovine adenovirus (BAdV) types have been sequenced, analysed and compared to the ITRs of other adenoviruses. The length of ITRs of the examined BAdVs ranged between 59 and 368 base pairs, thus the presently known longest adenovirus ITR sequence is from BAdV-10. The conserved motifs and characteristic sequence elements of the ITRs providing different binding sites for replicative proteins of viral and cellular origin seemed to be distributed according to the proposed genus classification of BAdVs. The ITRs of BAdV-10 share similarity with the members of the genus Mastadenovirus, while the ITRs of the other three sequenced serotypes (BAdV-4, 5 and strain Rus) which are candidate members of the genus Atadenovirus are very short and contain NFI and Sp1 binding sites only. The analysis of the new ITRs implied that the nucleotide sequence of the so-called core origin is highly preserved within the mastadenovirus genus only.