Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Kerber, Romy; Rieger, Toni; Busch, Carola; Flatz, Lukas; Pinschewer, Daniel D.; Kümmerer, Beate M.; Günther, Stephan

doi:10.1128/jvi.05091-11

Cited by 28 publications

(37 citation statements)

References 43 publications

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“…Although NP is not essential for the catalytic activity of L itself ( Fig. 1), it is likely that NP is critical for activities not recapitulated by the initial steps of RNA synthesis initiation in vitro, including integrity of the full-length genome and possibly reduction of RNA secondary structure (29,30). Here we demonstrate that Z is a direct modulator of the viral RNA synthesis machinery.…”

Section: Discussionmentioning

confidence: 99%

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Kranzusch

Whelan

2011

Proc. Natl. Acad. Sci. U.S.A.

104

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Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.transcription factor | Machupo virus | matrix protein | negative-strand RNA virus | gene regulation T ranscription initiation is a fundamental focal point of gene expression regulation in all orders of life. Modulation of RNA synthesis affords an important response to stress, alterations in growth conditions, and environmental cues. Although initiation of RNA synthesis is often indirectly controlled through gene promoters and accessory signaling elements, regulation also occurs through factors directly interacting with and altering the catalytic potential of the RNA synthesis machinery itself (1). Studies with phage T7 and the T7 lysozyme repressor system have provided an important paradigm for control of viral RNA transcription (2, 3), but these processes remain a mystery for many eukaryotic viruses and medically relevant pathogens. Gene expression regulation is particularly critical for negative-sense RNA viruses of the family Arenaviridae, which must respond to input from the host environment to maintain a noncytolytic infection in their rodent reservoir (4). In stark contrast to this persistent infection, arenavirus infection in humans can lead to severe hemorrhagic fever disease and many arenaviruses represent important emerging pathogens (5). Intriguingly, recent evidence indicates that the balance between persistent and acute viral infection can be altered by a single mutation within the viral polymerase (6). Mechanistic insight into how arenaviruses tune gene expression and control viral RNA synthesis is required to further our understanding of the response of arenavirus infection to various host and cellular environments.Arenaviruses are the simplest of all hemorrhagic fever viruses, with only four viral proteins. The viral genome consists of two segments of single-stranded negative-sense RNA, where each segment encodes two proteins in an ambisense orientation. The small segment encodes for the viral glycoprotein, essential for entr...

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Section: Discussionmentioning

confidence: 99%

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Kranzusch

Whelan

2011

Proc. Natl. Acad. Sci. U.S.A.

104

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“…To date there is no direct evidence of reassortant bunyaviruses emerging due to co-infection in an animal. As for arenaviruses, despite reassortment occurring in vitro , the risk of recombining segments is low, possibly due to superinfection exclusion; and as yet no natural reassortants have been detected for the arenaviruses3940. The current study shows that viral transmission was not detected between NHP groups infected with segmented viruses of the same family (RVF/CCHFV and LASV/MACV) therefore minimizing the possibility for reassortment.…”

Section: Discussionmentioning

confidence: 59%

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory

Alimonti

Leung

Jones

et al. 2014

Sci Rep

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Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route.

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“…Intriguingly, a previous study reported coimmunoprecipitation (co-IP) of L-NP in the absence of arenavirus-specific RNA with the use of a vaccinia virus system (MVAT7pol)-based expression system for L and NP (21). These apparently conflicting findings can be explained considering that the MVA-T7pol-based expression system results in hyperphysiological levels of L and NP that may overcome the requirement for virus-specific RNA.…”

Section: Mg Igrmentioning

confidence: 56%

Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template

Iwasaki

Ngo

Cubitt

et al. 2015

J Virol

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In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the two trans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5= and 3= termini of the viral genome. Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus (JUNV), cause hemorrhagic fever disease in humans and pose important public health concerns in regions where the viruses are endemic (1-6). Moreover, evidence indicates that the worldwidedistributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical relevance especially in cases of congenital and immunocompromised-individual infections (7-12). Arenaviruses are enveloped viruses with a bisegmented, negative-strand RNA genome. Each of the two RNA segments, S and L, uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region (IGR) (13). The S segment encodes the nucleoprotein (NP) and the surface glycoprotein precursor (GPC), whereas the L segment encodes the matrix (Z protein) and the RNA-dependent RNA polymerase (L protein). We reported that the IGR of LCMV is necessary for transcription termination and production of infectious progeny, indicating that noncoding viral RNA sequences play different functions during the arenavirus life cycle (14). In this work, we further extended our studies on the functional roles played by noncoding arenaviral RNA sequences by examining the contribution of these sequences to the interaction between the nucleocapsid template (NP-RNA) and L polymerase required for formation of a functional virus ribonucleoprotein (vRNP) responsible for directing RNA replication and gene transcription of the arenavirus genome.To examine whether virus-specific RNA sequences affected L-NP interaction, we used our LCMV minigenome (MG) system (15, 16). We transfected human embryonic kidney (HEK) 293T cells with NP-and L-expressing as well as T7 RNA polymerase (T7 pol)-expressing plasmids together with or without a plasmid that directed T7 pol-mediated intracellular synthesis of an LCMV MG RNA containing the chloramphenicol acetyltransferase (CAT) gene in the NP locus (pT7-MG) (15-18) and examined L-NP interaction by use of pulldown (PD) assays (Fig. 1). To facilitate PD assays and protein detection, we used tagged versions of NP and L that we had confirmed to be functionally active in the MG rescue assay (Fig. 1F and G), although FLAG-tagged L appeared to have some overall reduced activity in this assay. In the presence of MG RNA, L protein was readily detected by Western blotting (WB) in the complex PD by NP (Fig. 1B). In contrast, in the absence of MG RNA, the complex PD by NP showed dramatically reduced levels of L protein (Fig. 1B). Likewise, NP was efficiently pulled down with L protein in the presence, but not in the absence, of viru...

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Cross-Species Analysis of the Replication Complex of Old World Arenaviruses Reveals Two Nucleoprotein Sites Involved in L Protein Function

Cited by 28 publications

References 43 publications

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory

Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template

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