Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity

c Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.

Section: Figmentioning

confidence: 99%

Identification of a Role for Nucleolin in Rabies Virus Infection

Oksayan

Nikolić

David

et al. 2015

“…Since type I IFN signaling is important for protection against infection, it is not surprising that the RABV itself dedicates substantial resources to blocking type I IFN signaling. Both the N and P genes of RABV express products that interfere with type I IFN signaling (50,51), and mutation of P can attenuate the virus (52). The blockade of type I IFN by RABV is a "leaky" process, however, and the host response is able to impede replication and spread to some degree despite this immunoevasion strategy (48).…”

Section: Fig 10mentioning

confidence: 99%

Expression of Interferon Gamma by a Recombinant Rabies Virus Strongly Attenuates the Pathogenicity of the Virus via Induction of Type I Interferon

Barkhouse

Garcia

Bongiorno

et al. 2015

Previous animal model experiments have shown a correlation between interferon gamma (IFN-␥) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-␥ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-␥ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBN␥. Morbidity and mortality were monitored in mice infected intranasally with SPBN␥ or SPBN(؊) control virus to determine the degree of attenuation caused by the expression of IFN-␥. Incorporation of IFN-␥ into the rabies virus genome highly attenuated the virus. SPBN␥ has a 50% lethal dose (LD 50) more than 100-fold greater than SPBN(؊). In vitro and in vivo mouse experiments show that SPBN␥ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR ؊/؊ ) cannot control the SPBN␥ infection and rapidly die. These data suggest that IFN-␥ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-␥ production in the CNS prior to the appearance of virus-neutralizing antibodies.Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-␥ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-␥ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-␥ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus. R abies virus (RABV) is the type species of the Lyssavirus genus in the Rhabdoviridae family. Its small, negative-stranded RNA genome contains only five true genes (1, 2). Although relatively simple, this zoonotic virus has a devastating impact worldwide. The majority of human rabies deaths occur in children in the developing world, and it is estimated that at least 55,000 humans die of rabies each year in Africa and Asia alone (3).Although RABV infection historically has been viewed as a death sentence once the virus reaches the brain, there is a small but growing number of humans who have survived rabies even though the virus entered the brain (4, 5). Due to such cases and to research using animal models of RABV infection (6-8), many believe that the immune system may be cap...

“…Specifically, this protein plays an essential role in viral RNA synthesis as a cofactor of viral RNA-dependent RNA polymerase (L protein) by bridging nucleoprotein (N protein), which directly binds to viral genomic RNA, and L protein in the ribonucleoprotein complex (reviewed in reference 3). In addition, P protein functions to antagonize the type I interferon (IFN)-mediated antiviral responses by inhibiting both signaling pathways for IFN induction and response (4)(5)(6)(7)(8)(9)(10)(11)(12). P protein suppresses activation of interferon regulatory factor 3 (IRF-3), which is an important transcription factor for IFN induction (5,8).…”

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confidence: 99%

“…In addition, P protein functions to antagonize the type I interferon (IFN)-mediated antiviral responses by inhibiting both signaling pathways for IFN induction and response (4)(5)(6)(7)(8)(9)(10)(11)(12). P protein suppresses activation of interferon regulatory factor 3 (IRF-3), which is an important transcription factor for IFN induction (5,8). Also, P protein binds to the transcriptional factors signal transducers and activator of transcription 1 (STAT1) and STAT2, which play a key role in the IFN response by activating expression of IFN-stimulated genes (ISGs), and inhibits their nuclear translocation and DNA binding (6,10,11).…”

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confidence: 99%

“…Notably, all of the RABV P protein isoforms (P1 and tPs) retain a functionally important domain for inhibition of IRF-3 activation (amino acids 176 to 181 in P1) (8) as well as the STAT1-binding domain (amino acids 267 to 297 in P1) (10), implying that tPs have activity to antagonize the host IFN system. In fact, by using a recombinant protein expression system, it was previously demonstrated that P3 inhibits nuclear translocation and DNA binding of STATs (11,17).…”

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confidence: 99%

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Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis

Okada

Ito

Yamaoka

et al. 2016

Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)⌬P2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)⌬P2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)⌬P2-5 caused significantly lower morbidity and mortality rates in mice than did CE ( Rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, is a zoonotic agent that causes a lethal neurological disease in various mammal species, including humans. After transmission via a bite wound caused by an infected animal, RABV infects peripheral nerves and then spreads to and in the central nervous system (CNS), resulting in severe neurological symptoms with a high case fatality rate of almost 100% (reviewed in reference 1). Due to the absence of an effective cure and insufficient provision of postexposure prophylaxis, approximately 59,000 people die from rabies every year, mainly in developing countries (2). To establish an effective cure and also to develop a novel prophylaxis approach for rabies, it is necessary to understand the molecular mechanisms of the pathogenesis, including immune evasion, of RABV.The phosphoprotein (P protein) of RABV is a multifunctional protein that is indispensable not only for viral replication but also for evasion of host innate immunity. Specifically, this protein plays an essential role in viral RNA synthesis as a cofactor of viral RNA-dependent RNA polymerase (L protein) by bridging nucleoprotein (N protein), which directly binds to viral genomic RNA, and L protein in the ribonucleoprotein complex (reviewed in reference 3). In addition, P protein functions to antagonize the type I interferon (IFN)-mediated antiviral responses by inhibiting both signaling pathways for IFN induction and response (4-12). P protein suppresses activation of interferon regulatory factor 3 (IRF-3), which is an important transcription factor for IFN induction (5, 8). Also, P protein binds to the transcriptional factors signal transducers and activator of transcription 1 (STAT1) and STAT2, which play a key role in the IFN response by activating expression of IFN-stimulated genes (ISGs), and inhibits their nuclear translocation and DNA binding (6,10,11).In RABV-infected cells, mRNA of the P gene is translated from five in-frame start codons by a ribosomal leaky scanning mechanism, resulting in expression of full-length P protein (P1; 297 amino acids) and also less abundant expression of N-terminally truncated P proteins (t...