Eric B. Carstens

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015)

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Consensus statement: Virus taxonomy in the age of metagenomics

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus

The nucleotide sequence of the 60.1- to 65.5-m.u. region of Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined. Seven large open reading frames were identified. Two open reading frames potentially encoding gene products of 143 and 38 kDa were found in the counterclockwise direction upstream of the p6.9 gene. Four additional open reading frames were found in the opposite direction. Analysis of the predicted amino acid sequence of the 143-kDa gene revealed a potential leucine zipper motif, a putative nuclear localization signal, and seven amino acid motifs previously identified in a number of proteins involved in NTP binding and DNA/RNA unwinding. The mutation in a DNA replication defective temperature-sensitive mutant was fine mapped to the carboxy terminus of the ORF1(p143) gene. Sequence analysis of the mutation site identified a single base change of a guanine to an adenine, resulting in the substitution of a methionine for valine. This mutation resides seven amino acids downstream of the putative NTP-binding motif of the ORF1(p143) gene product and results in a DNA negative mutant. Together these data strongly suggest that the ORF1(p143) gene product is a baculovirus helicase.

Phenotypic characterization and physical mapping of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus defective in DNA synthesis

A ts mutant of Autographa californica nuclear polyhedrosis virus (AcMNPV), ts8, was shown to be defective in viral DNA synthesis at the nonpermissive temperature. Ts8-infected cells synthesized only early viral polypeptides at the nonpermissive temperature, and in contrast to wild-type (WT)-infected cells, showed no inhibition of host cell protein synthesis. The effect of the mutation on viral DNA synthesis was not immediately reversed after shifting infected cells down from the nonpermissive temperature to the permissive temperature; rather, a delay of several hours occurred before viral DNA synthesis was detected. The rate of accumulation of viral DNA in ts8-infected cells failed to increase after a shift from the permissive temperature to the nonpermissive temperature. This indicated that the ts8 mutation was involved in the synthesis of proteins required for viral DNA synthesis. The mutation was mapped by marker rescue to the region lying between 60.1 and 62.0% of the AcMNPV physical map.

Identification and nucleotide sequence of the regions of Autographa californica nuclear polyhedrosis virus genome carrying insertion elements derived from Spodoptera frugiperda

A morphology mutant of the baculovirus Autographa californica nuclear polyhedrosis virus called M5 was previously shown to synthesize two size classes of viral DNA, one of which had a deletion of 42% of the genome. It was hypothesized that the presence of the smaller M5 circular DNA resulted from the specific deletion of the region located between two sites carrying short DNA insertions. These sites have now been identified by DNA-DNA hybridization using cloned EcoRI fragments of M5 DNA. One cloned M5 EcoRI fragment was found to correspond to the deletion junction fragment where sequences from the insertion site in the 2.6-map unit region were covalently linked to sequences from the insertion site in the 46-map unit region. The 2.6- and 46-map unit regions of Wt AcMNPV DNA were sequenced. Potential long open reading frames which would be disrupted by the M5 inserts were detected. Nucleotide sequence analysis of the same regions of M5 DNA revealed the presence of almost identical inserts of 290 bp. The primary sequence of the inserts revealed characteristics similar to the termini of transposons. Hybridization studies suggested that the inserts were derived from repetitive elements of the Spodoptera frugiperda host cell genome.

Mapping early transcription products of Autographa californica nuclear polyhedrosis virus

The regions of the AcMNPV genome represented as cytoplasmic transcripts early after infection of Spodoptera frugiperda cells prior to and encompassing the initiation of DNA replication were mapped. In vivo(32)P pulse-labeled cytoplasmic RNA from infected cells at various times postinfection was used to probe Southern blots of cloned EcoRI AcMNPV DNA fragments. At the earliest time point studied (0.5 - 2.5 hr p.i.) transcripts represented a large proportion of the genome although specific regions were predominantly transcribed (EcoRI A, C, F, J, O, Q, S, and T). After the onset of viral DNA synthesis predominant cytoplasmic transcripts represented more dispersed areas of the genome, particularly those regions which were only weakly transcribed earlier after infection. Genomic regions containing major late genes were not predominantly transcribed by 7.5 hr p.i.

Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143.

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 3 (LEF-3) is an essential protein for DNA replication in transient assays. P143, a large DNA-binding protein with DNA-unwinding activity, is also essential for viral DNA replication in vivo. Both LEF-3 and P143 are found in the nucleus of AcMNPV-infected cells, but only LEF-3 localizes to the nucleus when expressed in transfected cells on its own from a plasmid expression vector. P143 requires LEF-3 as a transporter to enter the nucleus. To investigate the possibility that LEF-3 carries a nuclear localization signal domain, we constructed a series of LEF-3 deletion mutants and examined the intracellular localization of the products in plasmid-transfected cells. We discovered that the N-terminal 56 amino acid residues of LEF-3 were sufficient for nuclear localization and that this domain, when fused with either the green fluorescent protein reporter gene or P143, was able to direct these proteins to the nucleus. Transient DNA replication assays demonstrated that fusing the LEF-3 nuclear localization signal domain to P143 did not alter the function of P143 in supporting DNA replication but was not sufficient to substitute for whole LEF-3. These data show that although one role for LEF-3 during virus infection is to transport P143 to the nucleus, LEF-3 performs other essential replication functions once inside the nucleus.

Recently agreed changes to the International Code of Virus Classification and Nomenclature

The International Committee on Taxonomy of Viruses (ICTV; http://www.ictvonline.org/) is a committee of Virology Division of the International Union of Microbiological Societies, and its operation is governed by Statutes agreed with Virology Division. The classification and nomenclature of viruses is then subject to rules formalized into a Code (required by Statute 8.1). The need for a review of the Statutes and Code became clear during editorial work involved in production of the Ninth ICTV Report [1]. Changes to these documents require the approval of the ICTV Executive Committee (EC) and the full ICTV membership. Changes to the Statutes also require the agreement of Virology Division. The changes described in this article were discussed and agreed by the ICTV EC over a period of more than two years. Notification of a ballot was sent via email on 14 January 2013 to the 165 members of ICTV, namely the ICTV EC Members, Life Members, ICTV Subcommittee Members and ICTV National Representatives. Members were then requested to vote on whether or not to ratify the proposals (voting closed on 14 February 2013). The return rate of votes was approximately 41%, and all proposed changes were accepted, mostly unanimously. The only significant exception was the proposed change to the species definition in Rule 3.21 (Rule 3.20 in the latest version of the code) which was approved by 45:21 with two abstentions. In this article, we present the new version of the Code, highlighted in bold to show those parts that have recently been changed. Those changes are then explained in the sections that follow. It should be noted that some rules were deleted and that some renumbering of sections has therefore taken place. In recent years, the Code has been published with some explanatory Comments from the EC, and the opportunity has been taken to revise and update these where necessary. The changes to the Statutes are the subject of a separate article [2].

Characterization of the Interaction between P143 and LEF-3 from Two Different Baculovirus Species: Choristoneura fumiferana Nucleopolyhedrovirus LEF-3 Can Complement Autographa californica Nucleopolyhedrovirus LEF-3 in Supporting DNA Replication

ABSTRACT The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.

Analysis of polyhedra morphology mutants of Autographa californica nuclear polyhedrosis virus: molecular and ultrastructural features

Two new mutants of Autographa californica multiple nuclear polyhedrosis virus affected in the morphogenesis of their polyhedra, designated M276 and M934, were investigated. Marker transfer experiments demonstrated that the observed phenotype was due exclusively to alterations in the polyhedrin gene. M276 contained a 229 base insertion near the carboxyl terminus coding region which resulted in synthesis of a truncated protein; M934 had a point mutation substituting phenylalanine for leucine at amino acid 183. Both mutations occurred in highly conserved regions of the protein and prevented the occlusion of virus particles, but did not affect targeting for the intranuclear ring zone. M276 was distinct in that it had prominent cytosolic condensations of polyhedrin, although these were probably due to a decreased protein solubility. M934 polyhedrin condensations associated prematurely with calyx material such that it became incorporated into the condensation rather than at the surface. Results confirm that occlusion size and shape are features inherent to the polyhedrin protein, and suggest that polyhedrin conformation may help regulate the occlusion process.