Peter Tijssen

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 family Parvoviridae

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of “parvovirus” and “densovirus” to denote multiple taxon levels.

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.

Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 Å resolution

BACKGROUND Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.

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.

Multiple Pathways Involved in Porcine Parvovirus Cellular Entry and Trafficking toward the Nucleus

ABSTRACT Porcine parvovirus (PPV) is a major cause of reproductive failure in swine. The mechanisms implicated in the first steps of infection that lead to the delivery of the PPV genome to the nucleus are poorly understood. In the present work, a panel of chemical inhibitors was used to dissect the cellular mechanisms involved in establishing a PPV infection. The results demonstrated that following binding to sialic acids on cell surface glycoproteins, the virus used both clathrin-mediated endocytosis and macropinocytosis pathways to gain access into cells. Virus obtained from infected cells was present either as isolated particles or as aggregates, and these two forms could be separated by low-speed centrifugation. Isolated and purified particles strongly preferred entry by clathrin-mediated endocytosis, whereas aggregates clearly favored macropinocytosis. Subsequent endosomal acidification and traffic to the late endosomes were also shown to be essential for infection. The microtubule network was found to be important during the first 10 h of infection, whereas an intact actin network was required for almost the whole viral cycle. Proteasome processing was found to be essential, and capsid proteins were ubiquitinated relatively early during infection. Taken together, these results provided new insights into the first steps of PPV infection, including the use of alternative entry pathways, unique among members of this viral family.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

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.