A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

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Theiler's Virus L* Protein Is Targeted to the Mitochondrial Outer Membrane

Journal of Virology ◽

10.1128/jvi.02023-10 ◽

2011 ◽

Vol 85 (7) ◽

pp. 3690-3694 ◽

Cited By ~ 7

Author(s):

F. Sorgeloos ◽

D. Vertommen ◽

M. H. Rider ◽

T. Michiels

Keyword(s):

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Theiler's Virus ◽

L Protein

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Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.00573-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 4

Author(s):

Melissa Drappier ◽

Fred R. Opperdoes ◽

Thomas Michiels

Keyword(s):

Encephalitis Virus ◽

Species Specificity ◽

Functional Assay ◽

Rnase L ◽

Theiler's Murine Encephalomyelitis Virus ◽

L Protein ◽

Theiler’S Murine Encephalomyelitis Virus ◽

Human Rnase ◽

Encephalomyelitis Virus ◽

Species Specific

ABSTRACT Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV. IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L, indicating that this virus originates from mice.

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The Leader Protein of Theiler's Virus Inhibits Immediate-Early Alpha/Beta Interferon Production

Journal of Virology ◽

10.1128/jvi.75.17.7811-7817.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7811-7817 ◽

Cited By ~ 92

Author(s):

Vincent van Pesch ◽

Olivier van Eyll ◽

Thomas Michiels

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Zinc Binding ◽

Binding Motif ◽

Theiler's Virus ◽

L Protein ◽

Beta Interferon ◽

The Central Nervous System ◽

Alpha Beta ◽

Zinc Binding Motif

ABSTRACT Theiler's virus is a picornavirus responsible for a persistent infection of the central nervous system of the mouse, leading to a chronic demyelinating disease considered to be a model for multiple sclerosis. The leader (L) protein encoded by Theiler's virus is a 76-amino-acid-long peptide containing a zinc-binding motif. This motif is conserved in the L proteins of all cardioviruses, including encephalomyocarditis virus. The L protein of Theiler's virus was suggested to interfere with the alpha/beta interferon (IFN-α/β) response (W.-P. Kong, G. D. Ghadge, and R. P. Roos, Proc. Natl. Acad. Sci. USA 91:1796–1800, 1994). We show that expression of the L protein indeed inhibits the production of alpha/beta interferon by infected L929 cells. The L protein specifically inhibits the transcription of the IFN-α4 and IFN-β genes, which are known to be activated early in response to viral infection. Mutation of the zinc finger was sufficient to block the anti-interferon activity, outlining the importance of this motif in the L protein function. In agreement with the anti-interferon role of the L protein, a virus bearing a mutation in the zinc-binding motif was dramatically impaired in its ability to persist in the central nervous system of SJL/J mice.

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Influence of the Theiler's Virus L∗ Protein on Macrophage Infection, Viral Persistence, and Neurovirulence

Journal of Virology ◽

10.1128/jvi.74.19.9071-9077.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9071-9077 ◽

Cited By ~ 32

Author(s):

Olivier van Eyll ◽

Thomas Michiels

Keyword(s):

Viral Persistence ◽

Initiation Codon ◽

Divergent Evolution ◽

Theiler's Virus ◽

Macrophage Infection ◽

Reading Frame ◽

L Protein ◽

Related Clone ◽

Weak Influence

ABSTRACT The genome of picornaviruses contains a large open reading frame (ORF) translated as a precursor polypeptide that is processed to yield all the proteins necessary for the viral life cycle. In persistent but not in neurovirulent strains of Theiler's virus, an overlapping ORF encodes an additional 18-kDa protein called L∗. We confirmed previous work showing that the L∗ ORF of persistent strains facilitates the infection of macrophage cell lines, and we present evidence that this effect is due to the L∗ protein itself rather than to competition for the translation of the two overlapping ORFs. The introduction of an AUG codon to restore the L∗ ORF of the neurovirulent GDVII strain also enhanced the infection of macrophages, in spite of the divergent evolution of this protein. The presence or the absence of the L∗ AUG initiation codon had only a weak influence on the neurovirulence of the GDVII strain and on the persistence of the DA1 strain. The results obtained with DA1 in vivo contrast with the results reported previously for DAFL3, another molecular clone of the same virus strain, where the AUG-to-ACG mutation of the L∗ initiation codon totally blocked viral persistence (G. D. Ghadge, L. Ma, S. Sato, J. Kim, and R. P. Roos, J. Virol. 72:8605–8612, 1998). Thus, a factor that is critical for the persistence of a given clone of Theiler's virus is dispensable for the persistence of a closely related clone, indicating that different adjustments in the expression of persistence determinants occur in related viral strains.

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A Theiler’s virus BeAn strain shows a persistent profile of BHK-21 cell infection as determined by genome detection using real-time RT-PCR and expression of L* protein

Archives of Virology ◽

10.1007/s00705-012-1434-4 ◽

2012 ◽

Vol 157 (12) ◽

pp. 2437-2440

Author(s):

Alvaro Jorge Velloso ◽

Haroldo Cid da Silva Junior ◽

Eneida Almeida Santos ◽

Eliane Barbosa Baroni ◽

Marcia Terezinha Baroni de Moraes

Keyword(s):

Real Time ◽

Theiler's Virus ◽

Rt Pcr ◽

Cell Infection ◽

L Protein

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Non-AUG-Initiated Internal Translation of the L* Protein of Theiler's Virus and Importance of This Protein for Viral Persistence

Journal of Virology ◽

10.1128/jvi.76.21.10665-10673.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10665-10673 ◽

Cited By ~ 51

Author(s):

Olivier van Eyll ◽

Thomas Michiels

Keyword(s):

Demyelinating Disease ◽

Open Reading Frame ◽

Initiation Codon ◽

Theiler's Virus ◽

Entry Site ◽

Internal Ribosome Entry ◽

Reading Frame ◽

L Protein ◽

The Central Nervous System ◽

Internal Translation

ABSTRACT Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either acute encephalitis or persistent demyelinating disease. Persistent strains of Theiler's virus (such as DA) produce an 18-kDa protein called L* from an open reading frame overlapping that encoding the viral polyprotein. Neurovirulent strains (such as GDVII) are thought not to produce the L* protein, as the alternative open reading frame of these strains starts with an ACG codon instead of an AUG codon. However, we observed that both persistent and neurovirulent strain derivatives can produce two forms of the L* protein through unusual type II internal ribosome entry site-mediated translation. A full-length 18-kDa protein can be expressed from an ACG or an AUG initiation codon, whereas an N-terminally truncated 15-kDa product can be translated from a downstream AUG initiation codon. The expression of the 18-kDa form is required for efficient persistence of DA virus derivatives in the central nervous system.

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