Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-08-11
2003-01-07
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S006120, C435S007100, C435S007400, C435S007710, C435S007800, C435S007920, C435S007940, C435S007930, C435S007950, C435S008000
Reexamination Certificate
active
06503703
ABSTRACT:
1. INTRODUCTION
The present invention relates to the identification of new cellular targets for viral intervention, the identification of antiviral compounds that act on the new targets, and the therapeutic use of such antiviral compounds.
2. BACKGROUND OF THE INVENTION
Replication of viruses can induce drastic changes in the infected host cell metabolism. The analysis of the replication cycle of viruses by molecular biological techniques has facilitated the identification and study of viral gene products that modulate and affect cellular functions (Knipe, 1996, in Fields Virology-1996, Fields, et al., eds., Raven Publishers, Philadelphia, Pa., p. 273-299).
2.1. Influenza a Viral Gene Products that Modulate host Cellular Functions
Influenza A virus is a negative strand RNA virus belonging to the orthomyxovinis family. The genome of the virus consists of 8 segments and encodes 10 polypeptides. Experimental evidence generated in the laboratory of Scholtissek indicates that the nucleoprotein (NP) is a major determinant of species specificity of influenza viruses (Scholtissek, et al., 1985, Virology 147: 287-294).
2.1.1. NP PROTEIN
Transcription and replication of influenza virus RNA takes place in the nucleus of the infected cell. Transcription and replication of influenza virus RNA requires four virus encoded proteins: the NP and the three components of the viral RNA-dependent RNA polymerase, PB1, PB2 and PA (Huang, et al., 1990, J. Virol. 64: 5669-5673). The NP is the major structural component of the virion that interacts with genomic RNA, and is required for antitermination during RNA synthesis (Beaton & Krug, 1986, Proc. Natl. Acad. Sci. USA 83:6282-6286). NP is also required for elongation of RNA chains (Shapiro & Krug, 1988, J. Virol. 62: 2285-2290) but not for initiation (Honda, et al., 1988, J. Biochem. 104: 1021-1026).
Phylogenetic analysis divides NP genes into two families: one containing NPs predominantly of avian origin, and one containing those of human origin (Bean, 1984, Virology 133: 438-442; Buckler-White & Murphy, 1986, Virology 155: 345-355; Gammelin, et al., 1989, Virology 170: 71-80; Scholtissek, et al., 1985, Virology 147: 287-294). The human virus A/HK/1/68 and viruses having genetically related NPs cannot rescue mutants of the avian virus A/FPV/Rostock/1/34 (FPV) with temperature sensitive (ts) defects in the NP following double infection of chicken embryo fibroblasts (CEF) at 40° C. (Scholtissek, et al., 1985, Virology 147: 287-294; Scholtissek, et al., 1978, Virology 91: 79-85). However, the human viruses that failed to rescue the ts mutants on CEF cells were able to do so on Madin-Darby canine kidney (MDCK) cells (Scholtissek, et al., 1978, Virology 91: 79-85). Additionally, A/HK/1/68 virus and A/FPV/Rostock/1/34 virus reassortants containing the A/HK/1/68 virus-derived NP replicate in MDBK cells (bovine kidney) but not in CEFs (Scholtissek, et al., 1978, Virology 91: 79-85). The host-specific rescue of FPV ts mutants and the host restriction of A/HK/1/68 virus reassortants suggest that a factor(s) of host origin, which differs between mammalian and avian cells, is responsible for this phenomenon, and that this factor may interact with the influenza A virus NP. However, no host protein(s) that interacts with NP during infection has previously been identified or characterized.
2.1.2. NS1 PROTEIN
The NS1 protein of influenza A viruses is known to modulate and affect cellular functions. The NS1 is the only non-structural protein of the virus and is abundantly expressed in infected cells (Lazarowitz, et al., 1971, Virology 46: 830-843).
Several regulatory finctions of the NS1 protein have been identified. The NS1 protein may influence multiple steps of gene expression including pre-mRNA splicing (Fortes, et al., 1994, EMBO J. 13: 704-712; Lu, et al., 1994, Genes Dev. 8: 1817-1828), nucleo-cytoplasmic transport of poly(A)-RNA (Fortes, et al., 1994, EMBO J. 13: 704-712; Qiu, Y., et al., 1994, J. Virology 68: 2425-2432) and translation (De La Luna, S., et al., 1995, J. Virol. 69: 2427-2433; Enami, K., et al., 1994, J. Virol. 68: 1423-1427). In addition, NS1 can block the activation of the double-stranded RNA (dsRNA) activated protein kinase (PKR), presumably due to its dsRNA binding activity (Lu, et al., 1995, Virology 214: 222-228). The activation of PKR results in a downregulation of translation and is part of the cellular antiviral defense mechanisms The NS1 protein may counteract this cellular response in order to synthesize high levels of viral proteins in the infected cell (Lu, et al., 1995, Virology 214: 222-228). These pleiotropic effects may singly or combined provide the molecular basis for the role that the NS 1 protein plays in determining the host range and virulence of influenza virus strains (Shimizu K., et al., 1983, Virology 124: 35-44; Treanor, J. J., etal., 1989, Virology 171: 1-9).
Despite these studies examining the activities of NS1 and its interactions with various RNAs, little is known about the cellular factors that are recognized by the NS1 protein and that may therefore be central to NS1 functions.
2.2. RHABDOVIRUS GENE PRODUCTS THAT MODULATE HOST CELLULAR FUNCTIONS
Viruses belonging to the Rhabdoviridae family cause disease in a wide variety of species including vertebrates, invertebrates, and plants (Wagner & Rose, 1996, In Fields, et al., (eds.),
Fields Virology
, 3rd edition, Lippincott-Raven Publishers, Philadelphia, pp. 1121-1135). Two prototypic members of the Rhabdoviridae family include vesicular stomatitis virus (VSV; genus=vesiculovirus) and rabies virus (genus=lyssavirus). Like influenza A virus, rhabdoviruses possess a negative-strand RNA genome. Rhabdoviruses replicate exclusively in the cytoplasm of infected cells, and derive their lipid envelope via budding through the cytoplasmic membrane (for review see Wagner & Rose, 1996, In Fields, et al., (eds.),
Fundamental Virology
, 3rd edition, Lippincott-Raven Publishers, Philadelphia, pp. 1121-1135).
2.2.1. MATRIX (M) PROTEIN
Many aspects of the replication process of rhabdoviruses remain unclear. The major structural protein of rhabdoviruses, the matrix (M) protein, is thought to play a key role in viral assembly and release (Chong & Rose, 1993, J. Virol., 67, 407-414; Chong & Rose, 1994, J. Virol., 68, 441-447; Kaptur, et al., 1995, Virology, 206, 894-903; Lenard, 1996, Virology, 216, 289-298; Lyles, et al., 1992, J. Virol., 66, 349-358; McCreedy & Lyles, 1989, Virus Res., 14, 189-205; Mebatsion, et al., 1996, Cell, 84, 941-951; Pal & Wagner, 1987, In, Wagner (ed.),
The Rhabdoviruses.
Plenum, New York, pp. 75-128; Newcomb, et al., 1982, J. Virol., 41, 1055-1062; Zakowski, et al., 1981, Biochemistry, 20, 3902-3907). When the M protein of VSV is expressed in mammalian cells or a baculovirus system in the absence of any other viral protein, M protein is released from the cells in the form of lipid vesicles by budding through the cytoplasmic membrane (Justice, et al., 1995, J. Virol., 69, 3156-3160; Li, et al., 1993, J. Virol., 67, 4415-4420). The N-terminal portion of the VSV M protein has been shown to be important for membrane localization, and thus the budding process (Chong & Rose, 1993, J. Virol., 67, 407-414; Chong and Rose, 1994, J. Virol., 68, 441-447; Lenard and Vanderoef, 1990, J. Virol., 64, 3486-3491; Ye, et al., 1994, J. Virol. 68, 7386-7396; Zakowski and Wagner, 1980, J. Virol., 36, 93-102). The precise mechanism of how M is released from cells and the potential function(s) of host proteins in the budding process remain unclear.
The role of the M protein in rhabdoviral assembly has been compared to that of the gag protein in retroviral assembly (Lenard, 1996, Virology, 216, 289-298). The gag protein of Rous sarcoma virus (RSV) and the M protein of VSV share the ability to associate with the cytoplasmic membrane, and to bud from cells independent of other viral proteins (Justice, et al., 1995, J. Virol., 69, 3156-3160; Li, et al., 1993, J. Virol., 67, 4415-4420; Wills, et al., 1994, J. Virol., 68, 6605-6618). In addition to the membrane ass
Harty Ronald
O'Neill Robert
Palese Peter M
Mount Sinai School of Medicine of New York University
Pennie & Edmonds LLP
Qunhi Bao
Salimi Ali R.
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