An Arenavirus RING (Zinc-Binding) Protein Binds the Oncoprotein Promyelocyte Leukemia Protein (PML) and Relocates PML Nuclear Bodies to the Cytoplasm

ABSTRACT The promyelocytic leukemia protein (PML) forms nuclear bodies which are altered in some disease conditions. We report that the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV) influences the distribution of PML bodies. In cells infected with LCMV, the Z protein and PML form large bodies primarily in the cytoplasm. Transient transfection studies indicate that Z alone is sufficient to redistribute PML to the cytoplasm and that PML and Z colocalize. Coimmunoprecipitation studies show specific interaction between PML and Z proteins. A similar result was observed with a Z protein from another arenavirus, Lassa virus, suggesting that this is a general feature of the Arenaviridae. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML and does not need the PML RING or the nuclear localization signal to colocalize. The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants. The interaction between PML and Z may influence certain unique characteristics of arenavirus infection.

Download Full-text

Z Proteins of New World Arenaviruses Bind RIG-I and Interfere with Type I Interferon Induction

Journal of Virology ◽

10.1128/jvi.01362-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1785-1791 ◽

Cited By ~ 102

Author(s):

Lina Fan ◽

Thomas Briese ◽

W. Ian Lipkin

Keyword(s):

New World ◽

Rna Virus ◽

A549 Cells ◽

Lymphocytic Choriomeningitis ◽

Lassa Virus ◽

Type I ◽

Mrna Induction ◽

Machupo Virus ◽

Z Protein ◽

Transcriptional Induction

ABSTRACT The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-β) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-β response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-β mRNA induction in A549 cells in response to RNA bearing 5′ phosphates (5′pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-β.

Download Full-text

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

10.1101/2021.04.30.442226 ◽

2021 ◽

Author(s):

Jiao Guo ◽

Xiaoying Jia ◽

Yang Liu ◽

Junyuan Cao ◽

Gengfu Xiao ◽

...

Keyword(s):

Binding Affinity ◽

Rna Virus ◽

Treatment Options ◽

Hemorrhagic Fever ◽

Cell Types ◽

Lymphocytic Choriomeningitis ◽

Lassa Virus ◽

Viral Glycoprotein ◽

Mutant Proteins ◽

Alpha Dystroglycan

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever, for which there are limited treatment options. Successful LASV entry requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of GP1 mutant proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus residue (L84N, K88E, L10F, and H170S) reduced the binding affinity of GP1LASV for LAMP1. Moreover, all the mutations caused decreases in GPC-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, whereas L107F and H170S had only mild effects on infectivity. Notably, in LAMP1 knockout cells, all four mutants showed reduced pseudovirus infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order L84N > L107F > K88E > H170S.

Download Full-text

SAFA initiates innate immunity against cytoplasmic RNA virus SFTSV infection

PLoS Pathogens ◽

10.1371/journal.ppat.1010070 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010070

Author(s):

Bin-yan Liu ◽

Xue-jie Yu ◽

Chuan-min Zhou

Keyword(s):

Nucleocapsid Protein ◽

Inflammatory Responses ◽

Rna Virus ◽

Cytoplasmic Rna ◽

High Fatality Rate ◽

Signaling Axis ◽

Localization Signal ◽

Antiviral Mechanism ◽

Attachment Factor ◽

Severe Fever

Nuclear scaffold attachment factor A (SAFA) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS with a high fatality rate of up to 30%. It remains elusive whether and how cytoplasmic SFTSV can be sensed by the RNA sensor SAFA. Here, we demonstrated that SAFA was able to detect SFTSV infection and mediate antiviral interferon and inflammatory responses. Transcription and expression levels of SAFA were strikingly upregulated under SFTSV infection. SAFA was retained in the cytoplasm by interaction with SFTSV nucleocapsid protein (NP). Importantly, SFTSV genomic RNA was recognized by cytoplasmic SAFA, which recruited and promoted activation of the STING-TBK1 signaling axis against SFTSV infection. Of note, the nuclear localization signal (NLS) domain of SAFA was important for interaction with SFTSV NP and recognition of SFTSV RNA in the cytoplasm. In conclusion, our study reveals a novel antiviral mechanism in which SAFA functions as a novel cytoplasmic RNA sensor that directly recognizes RNA virus SFTSV and mediates an antiviral response.

Download Full-text

A Highly Conserved Leucine in Mammarenavirus Matrix Z Protein Is Required for Z Interaction with the Virus L Polymerase and Z Stability in Cells Harboring an Active Viral Ribonucleoprotein

Journal of Virology ◽

10.1128/jvi.02256-17 ◽

2018 ◽

Vol 92 (11) ◽

Cited By ~ 6

Author(s):

Masaharu Iwasaki ◽

Juan C. de la Torre

Keyword(s):

Hemorrhagic Fever ◽

Lymphocytic Choriomeningitis ◽

Lymphocytic Choriomeningitis Virus ◽

Negative Regulator ◽

Lassa Virus ◽

Membrane Composition ◽

Subcellular Compartment ◽

Rapid Degradation ◽

Wide Range ◽

Z Protein

ABSTRACT Mammarenaviruses cause chronic infections in their natural rodent hosts. Infected rodents shed infectious virus into excreta. Humans are infected through mucosal exposure to aerosols or direct contact of abraded skin with fomites, resulting in a wide range of manifestations from asymptomatic or mild febrile illness to severe life-threatening hemorrhagic fever. The mammarenavirus matrix Z protein has been shown to be a main driving force of virus budding and to act as a negative regulator of viral RNA synthesis. To gain a better understanding of how the Z protein exerts its several different functions, we investigated the interaction between Z and viral polymerase L protein using the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV). We found that in the presence of an active viral ribonucleoprotein (vRNP), the Z protein translocated from nonionic detergent-resistant, membrane-rich structures to a subcellular compartment with a different membrane composition susceptible to disruption by nonionic detergents. Alanine (A) substitution of a highly conserved leucine (L) at position 72 in LCMV Z protein abrogated Z-L interaction. The L72A mutation did not affect the stability or budding activity of Z when expressed alone, but in the presence of an active vRNP, mutation L72A promoted rapid degradation of Z via a proteasome- and lysosome-independent pathway. Accordingly, L72A mutation in the Z protein resulted in nonviable LCMV. Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required. IMPORTANCE Several mammarenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose important public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. The mammarenavirus matrix Z protein plays critical roles in different steps of the viral life cycle by interacting with viral and host cellular components. Here we report that alanine substitution of a highly conserved leucine residue, located at position 72 in LCMV Z protein, abrogated Z-L interaction. The L72A mutation did not affect Z budding activity but promoted its rapid degradation in the presence of an active viral ribonucleoprotein (vRNP). Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.

Download Full-text

Host-Driven Phosphorylation Appears to Regulate the Budding Activity of the Lassa Virus Matrix Protein

Pathogens ◽

10.3390/pathogens7040097 ◽

2018 ◽

Vol 7 (4) ◽

pp. 97 ◽

Cited By ~ 3

Author(s):

Christopher Ziegler ◽

Philip Eisenhauer ◽

Inessa Manuelyan ◽

Marion Weir ◽

Emily Bruce ◽

...

Keyword(s):

Protein Function ◽

Matrix Protein ◽

Rna Virus ◽

Hemorrhagic Fever ◽

Polymerase Activity ◽

Lassa Fever ◽

Lassa Virus ◽

Protein Z ◽

Western Africa ◽

Z Protein

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function.

Download Full-text

Two RING Finger Proteins, the Oncoprotein PML and the Arenavirus Z Protein, Colocalize with the Nuclear Fraction of the Ribosomal P Proteins

Journal of Virology ◽

10.1128/jvi.72.5.3819-3826.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3819-3826 ◽

Cited By ~ 78

Author(s):

Katherine L. B. Borden ◽

Elizabeth J. CampbellDwyer ◽

Graeme W. Carlile ◽

Mahmoud Djavani ◽

Maria S. Salvato

Keyword(s):

Nucleic Acid ◽

Excision Repair ◽

Rna Virus ◽

Nuclear Bodies ◽

Nuclear Fraction ◽

Nucleic Acid Binding ◽

Pml Bodies ◽

P Proteins ◽

Z Protein ◽

Ribosomal P

ABSTRACT The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.

Download Full-text

Screening and Identification of Lassa Virus Endonuclease-targeting Inhibitors from a Fragment-based Drug Development Library

10.1101/2021.07.09.451867 ◽

2021 ◽

Author(s):

Xiaohao Lan ◽

Yueli Zhang ◽

Yang Liu ◽

Jiao Guo ◽

Xiaoying Jia ◽

...

Keyword(s):

Drug Development ◽

Rna Virus ◽

Lymphocytic Choriomeningitis ◽

Lassa Virus ◽

Virus Infections ◽

Mrna Transcription ◽

Binding Ability ◽

Screening And Identification ◽

Negative Sense ◽

Severe Fever

Lassa virus (LASV) belongs to the Old World genus Mammarenavirus, family Arenaviridae, and order Bunyavirales. Arenavirus contains a segmented negative-sense RNA genome, which is in line with the bunyavirus and orthomyxoviruses. The segmented negative-sense RNA viruses utilize a cap-snatching strategy to provide primers cleavaged from the host capped mRNA for viral mRNA transcription. As a similar strategy and the conformational conservation shared with these viruses, the endonuclease (EN) would serve as an attractive target for developing broad-spectrum inhibitors. Using the LASV minigenome (MG) system, we screened a fragment-based drug development library and found three candidates (F1204, F1781, and F1597) inhibited MG activity. All three candidates also inhibited the prototype arenavirus Lymphocytic choriomeningitis virus (LCMV) MG activity. Furthermore, the investigation revealed that two benzotriazole compounds (F1204 and F1781) effectively inhibited authentic LCMV and severe fever with thrombocytopenia syndrome virus (SFTSV) infections. The combination of either compound with an arenavirus entry inhibitor had significant synergistic antiviral effects. Moreover, both F1204 and F1781 were found to exert the binding ability of LASV EN with binding affinity at the micromolar level. These findings provide a basis for developing benzotriazole compounds as potential candidates for the treatment of segmented negative-sense RNA virus infections.

Download Full-text