ABSTRACT
Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates â¼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.
Subject(s)
Gammainfluenzavirus/enzymology , Influenza A Virus, H5N1 Subtype/enzymology , Influenza B virus/enzymology , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza B virus/genetics , Gammainfluenzavirus/genetics , Lasers , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Nuclear Localization Signals/metabolism , Protein Interaction Domains and Motifs , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Ribonucleoproteins/metabolism , Scattering, Small Angle , Structure-Activity Relationship , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.
Subject(s)
Herpesvirus 4, Human/enzymology , Herpesvirus 4, Human/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Baculoviridae/genetics , Cell Line , DNA Helicases/genetics , DNA Helicases/metabolism , Genetic Vectors , Insecta , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , Viral Proteins/chemistryABSTRACT
Epstein-Barr virus (EBV) is a ubiquitous human pathogen which establishes life-long persistent infection in the large majority of the human population. During viral latency, the cellular machinery takes care of the replication of the viral episome. But EBV, as well as herpesviruses in general, codes for numerous enzymes required for lytic DNA replication which allow viral replication in resting cells. Recently, several tridimensional structures of these enzymes became available for EBV as well as for other herpesviruses so that structural information now exists for most of them. The replication process and the structures of the proteins involved in replication are reviewed in the light of potential drug development and of herpesvirus evolution. The structures of the proteins involved in lytic replication show the relationship between herpesviruses and tailed bacteriophages, furthermore they show that EBV proteins tend to be more complex than their counterparts in other organisms. In this review, we could show the phylogenetic position of the herpesvirus helicase close to the Dda helicases involved in initiation of replication of the caudovirales.
ABSTRACT
Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0 degrees C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.
