The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.

Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

This invention provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting the cell with an amount of a compound effective to inhibit binding of an HCV envelope glycoprotein to a DC-SIGN protein present on the surface of the cell, so as to thereby inhibit HCV infection of the cell susceptible to HCV infection. This infection provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting the cell with an amount of a compound effective to inhibit binding of an HCV envelope glycoprotein to a DC-SIGNR protein present on the surface of the cell, so as to thereby inhibit HCV infection of the cell susceptible to HCV infection. Compounds of the present invention inhibit HCV infection of cells susceptible to HCV infection. The compounds of the present invention preferably have specificity for preventing or inhibiting infection by HCV and do not inhibit infection by other viruses, such as HIV, that may utilize DC-SIGN or DC-SIGNR for infection. Moreover the compounds of the present invention preferably do not interfere or inhibit members of the immunoglobulin superfamily, in particular, the compounds do not interfere with ICAM-2 or ICAM-3 or with ICAM-2-ilke, or ICAM-3-like molecules.

Uses of dc-sign and dc-signr for inhibiting hepatitis c virus infection

Replication of poliovirus (PV) is restricted to a few sites, including the brain and spinal cord. However, this neurotropism is not conserved in cultured cells. Monkey kidney cells become susceptible to PV infection after cultivation in vitro, and cell lines of monolayer cultures from almost any tissue of primates are susceptible to PV infection. These observations suggest that cellular changes during cultivation are required for acquisition of susceptibility. The molecular basis for the cellular changes during this process is not known. We investigated the relationship between PV susceptibility and interferon (IFN) response in primary cultured kidney and liver cells derived from transgenic mice expressing human PV receptor and in several primate cell lines. Both kidneys and liver in vivo showed rapid IFN response within 6 h postinfection. However, monkey and mouse kidney cells in culture and primate cell lines, which were susceptible to PV, did not show such rapid response or showed no response at all. On the other hand, primary cultured liver cells, which were partially resistant to infection, showed rapid IFN induction. The loss of IFN inducibility in kidney cells was associated with a decrease in expression of IFN-stimulated genes involved in IFN response. Mouse kidney cells pretreated with a small dose of IFN, in turn, restored IFN inducibility and resistance to PV. These results strongly suggest that the cells in culture acquire PV susceptibility during the process of cultivation by losing rapid IFN response that has been normally maintained in extraneural tissues in vivo.

Role of the alpha/beta interferon response in the acquisition of susceptibility to poliovirus by kidney cells in culture.

Type 1, 2, and 3 vaccine-derived polioviruses were isolated from a sewage disposal plant located downstream of the Oyabe River in Toyama Prefecture, Japan, between October 1993 and September 1995. Neurovirulence was analyzed in 13 type 1 vaccine-derived strains, using mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC). Nine strains (69%) were estimated to have marked neurovirulence. Some of the neutralizing antigenic sites, temperature sensitivity, and plaque-forming ability of two virulent vaccine-derived poliovirus strains were similar to Mahoney strain. The neutralizing activity of human sera obtained after oral poliomyelitis vaccine (OPV) administration against one of the virulent vaccine-derived polioviruses was examined. Although all human sera showed sufficient neutralizing activity for the prevention of poliomyelitis by vaccine-derived poliovirus strains, a lower titer than that against Sabin type 1 strain was observed. Vaccination against virulent vaccine-derived poliovirus will be effective. However, the environmental presence of viruses that have properties similar to those Mahoney strain is a threat. The introduction of inactivated poliovirus vaccine (IPV), and well-maintained herd immunity, together with reinforced environmental surveillance is important for the final phase of the polio eradication program by the World Health Organization (WHO).

Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan.

Poliomyelitis, an acute disease of the central nervous system (CNS), is caused by poliovirus. Poliovirus is a human enterovirus belonging to the Picornaviridae, and is classified into three stable serotypes (types 1, 2, and 3). Poliovirus strains, except for type 2 virulent strains, infect only primates. The poliovirus infection is initiated by ingestion of a virus and its primary multiplication in the alimentary mucosa. The tonsils and Peyer’s patches are invaded early in the course of infection, and extensive viral multiplication occurs in these loci. From these primary sites, the virus moves into deep cervical and mesenteric lymph nodes, and then into the blood. When effective viremia is established, the virus spreads into the CNS, and then paralytic poliomyelitis occurs as a result of destruction of motor neurons in the brain and spinal cord through lytic viral replication.

Molecular Mechanisms of Poliovirus Pathogenesis

The present invention relates to a method of examining the neurovirulence of a polio virus, which comprises inoculating a polio virus into the spinal cord of a transgenic mouse comprising a gene for a polio virus receptor.

Method for examining neurovirulence of polio virus

A method of testing the neurotoxicity of poliovirus vaccine by inoculating the vaccine into the spinal cord of a transgenic nonprimatial vertebrate having poliovirus receptor genes introduced thereinto.

Method of testing neurotoxicity of poliovirus vaccine

step(s) ofthevirus infection cycle, different stepsoftheinfection cycle were examined fortemperature sensitivity. The results strongly suggested thatthegrowthrestriction observed at40°Cwas duetoreduced efficiency ofthe initiation processofvirus-specific RNA synthesis. Furthermore, this restriction appeared tooccur onlyon the synthesis ofpositive-strand RNA.Virus-specific RNA synthesis incrudereplication complexes was notaffected bythenonpermissive temperature of40°C. Invitro uridylylation ofVPgseemed tobetemperature sensitive onlyafter prolonged incubation at40°C. Theseresults indicate that a specific hostfactor(s) isinvolved inthe efficient initiation processofpositive-strand RNA synthesis ofpoliovirus andthatthehostfactor(s) is

Akio Nomoto

Papers

Molecular Mechanisms of Poliovirus Pathogenesis

Method for examining neurovirulence of polio virus

Method of testing neurotoxicity of poliovirus vaccine

MouseCellFactors forStrand-Specific Initiation ofPoliovirus RNA Synthesis