Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness

Abstract

e Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-␤) gene (Ifn-␤) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves. R abies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, infects almost all kinds of mammals, including humans, and causes a severe neurological disease with a high mortality rate of about 100% after a long and inconstant incubation period (usually 20 to 90 days in humans) (reviewed in reference 1). It is estimated that more than 55,000 people die of rabies every year, mainly in Asia and Africa (2), due to the absence of an effective cure and also the complexity and expensiveness of current postexposure prophylaxis, which requires medical treatment (i.e., rabies vaccination) five times over a period of 28 days. In order to develop both therapeutic and novel prophylaxis approaches for rabies, it is necessary to fully understand the pathogenesis of rabies.The pathogenesis of rabies essentially relies on viral spread to and in the nervous system of the infected individual (reviewed in reference 1). RABV secreted into saliva of a rabid animal is generally transmitted via a bite wound caused by the infected animal. After transmission, RABV infects peripheral nerves and then spreads to the central nervous system (CNS) via retrograde axonal transport, followed by active viral replication and spread in the CNS, culminating in severe neurological symptoms and lethal outcome. To date, studies...

Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis

“…We previously reported that RABV P protein has a key role in both neuroinvasiveness and neurovirulence (21,22). This is supported by experimental data showing that chimeric mutant CE(NiP), which has the P gene from virulent Nishigahara (Ni) in the genome of the attenuated variant Ni-CE, caused lethal infection in mice after both intramuscular (i.m.)…”

“…By using the same methods, we also obtained a recombinant CE(NiP)⌬P2-5 expressing firefly luciferase [CE(NiP)⌬P2-5-Luc] after inserting a PCR-amplified cDNA fragment of the luciferase gene into the G-L intergenic region of the above-described genome plasmid of CE(NiP)⌬P2-5 as previously reported (22,23). Details of the construction of the above-described genome plasmids will be provided by the authors on request.…”

“…CE(NiP) was previously generated by a reverse genetics approach (21). Recombinant CE(NiP) expressing firefly luciferase [CE(NiP)-Luc] was also generated in our previous study (22).…”

“…The infectious viruses in these stocks were titrated by focus assays on confluent NA cells as previously reported (22).…”

Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein

“…L protein functions were mostly predicted from studies on vesicular stomatitis virus. In recent years, reports about RABV were mainly focused on the viral G (5-7), P (8)(9)(10)(11)(12)(13)(14), M (15)(16)(17)(18), and N (9,(19)(20)(21)(22)(23)(24) proteins. However, the work on L protein of RABV has been relatively limited.…”