Lassa Virus Vaccine Candidate ML29 Generates Truncated Viral RNAs Which Contribute to Interfering Activity and Attenuation

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1-/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days).

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Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

Journal of Virology ◽

10.1128/jvi.01668-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2611-2622 ◽

Cited By ~ 42

Author(s):

Subash C. Das ◽

Debasis Panda ◽

Debasis Nayak ◽

Asit K. Pattnaik

Keyword(s):

Plasma Membrane ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Protein ◽

Dynamic Imaging ◽

Viral Envelope ◽

M Protein ◽

Recombinant Viruses ◽

Infected Cells ◽

Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

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Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA

Journal of Virology ◽

10.1128/jvi.00846-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9423-9431 ◽

Cited By ~ 10

Author(s):

Michael Zinke ◽

Sabine Kendl ◽

Katrin Singethan ◽

Markus Fehrholz ◽

Dajana Reuter ◽

...

Keyword(s):

Measles Virus ◽

Fluorescent Protein ◽

Lentiviral Vector ◽

Test System ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Persistently Infected ◽

Infected Cells ◽

Short Hairpin ◽

Nt2 Cells

ABSTRACT Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.

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Attenuated Replication of Lassa Virus Vaccine Candidate ML29 in STAT-1-/- Mice

Pathogens ◽

10.3390/pathogens8010009 ◽

2019 ◽

Vol 8 (1) ◽

pp. 9 ◽

Cited By ~ 5

Author(s):

Dylan Johnson ◽

Jenny Jokinen ◽

Igor Lukashevich

Keyword(s):

Vaccine Development ◽

Vaccine Candidate ◽

Small Animal ◽

Vero Cells ◽

Lassa Fever ◽

Effective Vaccine ◽

Cell Mediated Immunity ◽

Lassa Virus ◽

Cell Responses ◽

Fold Reduction

Lassa virus (LASV), a highly prevalent mammalian arenavirus endemic in West Africa, can cause Lassa fever (LF), which is responsible for thousands of deaths annually. LASV is transmitted to humans from naturally infected rodents. At present, there is not an effective vaccine nor treatment. The genetic diversity of LASV is the greatest challenge for vaccine development. The reassortant ML29 carrying the L segment from the nonpathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under current development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the worst outbreak on record in 2018. This study demonstrated that ML29 was more attenuated than MOPV in STAT1-/- mice, a small animal model of human LF and its sequelae. ML29 infection of these mice resulted in more than a thousand-fold reduction in viremia and viral load in tissues and strong LASV-specific adaptive T cell responses compared to MOPV-infected mice. Persistent infection of Vero cells with ML29 resulted in generation of interfering particles (IPs), which strongly interfered with the replication of LASV, MOPV and LCMV, the prototype of the Arenaviridae. ML29 IPs induced potent cell-mediated immunity and were fully attenuated in STAT1-/- mice. Formulation of ML29 with IPs will improve the breadth of the host’s immune responses and further contribute to development of a pan-LASV vaccine with full coverage meeting the WHO requirements.

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Infectious Lymphocytes in Mice Persistently Infected with Lymphocytic Choriomeningitis Virus

Zeitschrift für Naturforschung C ◽

10.1515/znc-1977-11-1228 ◽

1977 ◽

Vol 32 (11-12) ◽

pp. 1026-1028 ◽

Cited By ~ 5

Author(s):

Mircea Popescu ◽

Jürgen Löhler ◽

Fritz Lehmann-Grube

Keyword(s):

Persistent Infection ◽

Viral Antigen ◽

Infectious Virus ◽

Lymphocytic Choriomeningitis ◽

Lymphocytic Choriomeningitis Virus ◽

Mononuclear Phagocytes ◽

Infectious Process ◽

Persistently Infected ◽

Infected Cells ◽

Virus Carrier

Abstract During persistent infection of mice with the lymphocytic choriomeningitis (LCM) virus approximately 3% of leukocytes were found to contain viral antigen and to produce infectious virus. Morphologically, infected cells were shown not to be lymphoblasts and their numbers were not reduced by removal of mononuclear phagocytes. We conclude that in LCM virus carrier mice true lymphocytes participate in the infectious process.

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Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

Journal of General Virology ◽

10.1099/vir.0.19748-0 ◽

2004 ◽

Vol 85 (5) ◽

pp. 1181-1189 ◽

Cited By ~ 29

Author(s):

Sami K. J. Kukkonen ◽

Antti Vaheri ◽

Alexander Plyusnin

Keyword(s):

Matrix Protein ◽

Fluorescent Protein ◽

Protein Localization ◽

Expression System ◽

Peak Level ◽

Cell Fractionation ◽

L Protein ◽

System L ◽

Infected Cells ◽

In Cells

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 104 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

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Autophagy Promotes Infectious Particle Production of Mopeia and Lassa Viruses

Viruses ◽

10.3390/v11030293 ◽

2019 ◽

Vol 11 (3) ◽

pp. 293 ◽

Cited By ~ 6

Author(s):

Nicolas Baillet ◽

Sophie Krieger ◽

Alexandra Journeaux ◽

Valérie Caro ◽

Frédéric Tangy ◽

...

Keyword(s):

Matrix Protein ◽

Particle Production ◽

Defense Mechanism ◽

Viral Infections ◽

Hybrid Approach ◽

Hemorrhagic Fever ◽

Lassa Virus ◽

Infectious Particle ◽

Cellular Defense ◽

Infected Cells

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. We used a yeast two-hybrid approach to identify cell partners of MOPV and LASV Z matrix protein in which two autophagy adaptors were identified, NDP52 and TAX1BP1. Autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. Here, we demonstrate that autophagy is transiently induced by MOPV, but not LASV, in infected cells two days after infection. Impairment of the early steps of autophagy significantly decreased the production of MOPV and LASV infectious particles, whereas a blockade of the degradative steps impaired only MOPV infectious particle production. Our study provides insights into the role played by autophagy during MOPV and LASV infection and suggests that this process could partially explain their different pathogenicity.

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Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

Proceedings ◽

10.3390/proceedings2020050120 ◽

2020 ◽

Vol 50 (1) ◽

pp. 120

Author(s):

Juan-Vicente Bou ◽

Ron Geller ◽

Rafael Sanjuán

Keyword(s):

Genetic Variants ◽

Social Evolution ◽

Viral Population ◽

Multiplicity Of Infection ◽

Defective Interfering Particles ◽

Viral Genomes ◽

Viral Particles ◽

A Cell ◽

Infected Cells ◽

Population Sequence

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion.

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Protein analysis of defective interfering lymphocytic choriomeningitis virus and persistently infected cells

Virology ◽

10.1016/0042-6822(79)90107-7 ◽

1979 ◽

Vol 96 (2) ◽

pp. 503-515 ◽

Cited By ~ 38

Author(s):

Raymond M. Welsh ◽

Michael J. Buchmeier

Keyword(s):

Protein Analysis ◽

Lymphocytic Choriomeningitis ◽

Lymphocytic Choriomeningitis Virus ◽

Persistently Infected ◽

Infected Cells

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E3 Ligase ITCH Interacts with the Z Matrix Protein of Lassa and Mopeia Viruses and Is Required for the Release of Infectious Particles

Viruses ◽

10.3390/v12010049 ◽

2019 ◽

Vol 12 (1) ◽

pp. 49 ◽

Cited By ~ 2

Author(s):

Nicolas Baillet ◽

Sophie Krieger ◽

Xavier Carnec ◽

Mathieu Mateo ◽

Alexandra Journeaux ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Matrix Protein ◽

Virus Assembly ◽

Hemorrhagic Fever ◽

Lassa Fever ◽

Lymphocytic Choriomeningitis ◽

Lassa Virus ◽

Yeast Two Hybrid ◽

Ubiquitin Ligase Activity ◽

Two Hybrid

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.

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