- [Show abstract] [Hide abstract] ABSTRACT: The arenaviruses and hantaviruses are segmented genome RNA viruses that are hosted by rodents. Due to their association with rodents, they are globally widespread and can infect humans via direct or indirect routes of transmission, causing considerable human morbidity and mortality. Nevertheless, despite their obvious and emerging importance as pathogens, there are currently no effective antiviral drugs (except ribavirin which proved effective against Lassa virus) with which to treat humans infected by any of these viruses. The EU-funded VIZIER project (Comparative Structural Genomics of Viral Enzymes Involved in Replication) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the arenaviruses and bunyaviruses. This review highlights some of the major features of the arenaviruses and hantaviruses that have been investigated during recent years. After describing their classification and epidemiology, we review progress in understanding the genomics as well as the structure and function of replicative enzymes achieved under the VIZIER program and the development of new disease control strategies.
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Sequence Alignment of Viral Endonuclease Domains. A, Structure-based sequence alignment of NL1 with the two influenza PAN (3EBJ, 2W69), Tt1808 (1WDJ) and SdaI (2IXS), showing the structurally-conserved endonuclease motif (Highlighted in red). B, Sequence alignment of arenavirus NL1: Lymphocytic choriomeningitis virus (LCMV), Dandenong virus (DANV), Mopeia virus (MOPV), Morogoro virus (MORV), Mobala virus (MOBV), Ippy virus (IPPV), Lassa virus (LASV), Lujo Virus (LUJV), Parana virus (PARV), Pichinde virus (PICV), Allpahuayo virus (ALLV), Chapare virus (CHAV), Tamiami virus (TAMV), Whitewater Arroyo virus (WWAV), Bear Canyon virus (BCNV), Flexal virus (FLEV), Pirital virus (PIRV), Amapari virus (AMAV), Guanarito virus (GTOV), Cupixi virus (CPXV), Machupo virus (MACV), Junin virus (JUNV), Tacaribe virus (TCRV), Sabia virus (SABV), Oliveros virus (OLVV), Latino virus (LATV). Residues in a solid red background are strictly conserved. The blue point indicates the key active site residues. (4.29 MB TIF)
Dataset: Text S1
- [Show abstract] [Hide abstract] ABSTRACT: Author Summary The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Conservation of NL1 Between Arenaviruses and Bunyaviruses. A, Sequence alignment showing endonuclease motif (highlighted in red) of the NL1 domain from LCMV and the five genus of the Bunyaviridae families. Each genera is represented by two viruses: Orthobunyavirus: Human La Cross Virus (HLCV) and Bunyamwera virus (BUNV), Hantavirus: Hantaan virus (HANV) and Puumala virus (PUMV), Phlebovirus: Rift valley fever virus (RVFV) and Toscana virus (TOSV), Tospovirus: Tomato virus (TOMV) and Watermelon silver mottle virus (WTMV), Nairovirus: Crimean-Congo hemorrhagic fever virus (CCGV) and Dugbe virus (DUGV). Position of the start of the motif is labelled in blue for each virus. B, Based on secondary structure predictions topology diagrams were drawn for the NL1 domains from PUMV, RVFV, TOMV, HANV, CCGV. Colours are the same as in Figure 1D. N-terminal and C-terminal position are labelled with the aa position. The two red dots, the black dot and the green dot indicate the PD, E/D, and K residues, respectively, from the key active site. Principal conserved secondary structures are labelled as for LCMV NL1 domain. (8.97 MB TIF)
Dataset: Figure S3[Show abstract] [Hide abstract] ABSTRACT: Verification of L protein expression by immunoblotting. L protein used for analysis in replicon assay was tagged with HA tag, expressed under T7 promoter control in cells inoculated with modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA-T7), and detected in immunoblot using anti-HA antibody. MVA, cells inoculated with MVA-T7 but not transfected; neg. ctrl., L mutant containing a mutation in the catalytic site of the RdRp. (1.13 MB TIF)
- [Show abstract] [Hide abstract] ABSTRACT: In the treatment of HIV, the loose active site of the HIV-1 reverse transcriptase (RT) allows numerous nucleotide analogues to act as proviral DNA 'chain-terminators'. Acyclic nucleotide phosphonate analogues (ANPs) represent a particular class of nucleotide analogue that does not possess a ribose moiety. The structural basis for their substrate efficiency regarding viral DNA polymerases is poorly understood. Pre-steady-state kinetics on HIV-1 RT together with molecular modelling, were used to evaluate the relative characteristics of both the initial binding and incorporation into DNA of three different ANP diphosphates with progressively increasing steric demands on the acyclic linker: adefovir-diphosphate (DP), tenofovir-DP, and cidofovir-DP. The increase of steric demand in ANPs induced a proportional loss of the binding affinity to wild-type HIV-1 RT (Kd cidofovir-DP>Kd tenofovir-DP>Kd adefovir-DP approximately Kd dNTPs), consistent with the lack of HIV-1 inhibitory activity for cidofovir. We show that, starting from adefovir-DP, the steric constraints mainly map to Gln151, as its mutation to alanine provides cidofovir-DP sensitivity. Interactions between the Gln151 residue and the methyl group of tenofovir-DP further increase with the mutation Gln151Met, resulting in a specific discrimination and low-level resistance to tenofovir-DP. This alteration is the result of a dual decrease in the binding affinity (Kd) and the catalytic rate (k(pol)) of incorporation of tenofovir-DP. By contrast, the tenofovir resistance mutation K65R induces a broad 'k(pol)-dependent' nonspecific discrimination towards the three ANPs. Overall, our results show that the efficiency of ANPs to compete against natural nucleotides as substrates for RT is determined by their close interaction with specific amino acids such as Gln151 within the RT active site. These results should help us to map and predict ANP sensitivity determinants in cellular and viral DNA polymerase active sites for which the understanding of different ANP sensitivity patterns are of medical importance.
- [Show abstract] [Hide abstract] ABSTRACT: 9-[2-(Boranophosphonomethoxy)ethyl]adenine diphosphate (BH3-PMEApp) and (R)-9-[2-(boranophosphonomethoxy)propyl]adenine diphosphate (BH3-PMPApp), described here, represent the first nucleoside phosphonates modified on their α-phosphates that act as efficient substrates for the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). These analogues were synthesized and evaluated for their in vitro activity against wild-type (WT), K65R, and R72A RTs. BH3-PMEApp and BH3-PMPApp exhibit the same inhibition properties as their nonborane analogues on WT RT. However, K65R RT was found hypersensitive to BH3-PMEApp and as sensitive as WT RT to BH3-PMPApp. Moreover, the presence of the borane group restores incorporation of the analogue by R72A HIV RT, the latter being nearly inactive with regular nucleotides. The BH3-mediated suppression of HIV-1 RT resistance, formerly described with nucleoside 5′-(α-p-borano)-triphosphate analogues, is thus also conserved at the phosphonate level. The present results show that an α-phosphate modification is also possible and interesting for phosphonate analogues, a result that might find application in the search for a means to control HIV RT-mediated drug resistance.
- [Show abstract] [Hide abstract] ABSTRACT: Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Å resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.