Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-07-31
2002-06-04
Mosher, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100, C435S325000, C435S069700, C530S350000, C536S023400
Reexamination Certificate
active
06399354
ABSTRACT:
BACKGROUND OF THE INVENTION
Viruses play an important role in a person's health. Several viruses infect people for which no effective treatment plan exists. Examples of such viruses include herpesvirus, Human Immunodeficiency Virus (HIV) and the common cold. Finding effective anti-viral drugs depends on the ability to track the virus in a host or cell after the drug has been administered. Therefore, a need exists to be able to efficiently detect the presence or absence of a virus. A further need exists to develop screening methods for determining the efficacy of anti-viral agents or compounds. Another need exists to determine whether a cell is resistant to viral infection.
SUMMARY OF THE INVENTION
The invention embodies a fusion protein comprising a viral protein and a detectable protein. The viral protein and the detectable protein can be linked so that viral protein's native function is maintained. The detectable fusion protein can efficiently detect the presence, absence, or amount of viral DNA replication and, therefore, infection. The protein is thus a marker selected from a variety of viruses. Examples of such viruses are: the retrovirus (Human Immunodeficiency Virus (HIV)), the influenzavirus, the papillomavirus, the rhinovirus, and the herpesvirus (e.g., Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2), a Varicella-Zoster Virus, Epstein-Barr Virus, Cytomegalovirus, Human Herpesvirus-6, and Human Herpesvirus-7). In particular, a claimed invention embodies a viral protein from a Herpesvirus fused with a detectable protein (e.g., a fluorescent protein, or a protein that emits fluorescence upon excitation). An example of a fluorescent protein which can be used in the invention is a green fluorescent protein (GFP). In particular, a preferred embodiment utilizes the HSV ICP8 viral protein fused with a fluorescent protein, such as ICP8-GFP. In another embodiment, the invention pertains to antibodies which selectively bind to such a fusion protein, and plasmids or vectors that encode the fusion proteins, as described herein.
The invention also encompasses a virus that comprises a nucleic acid which expresses the claimed fusion protein. The recombinant virus which expresses the fusion protein is generally able to perform functions similar to a corresponding wild-type virus. The virus is preferably replication competent and in the case of the Herpesvirus able to form replication compartments, a “factory” where DNA synthesis takes place and possibly where virions are assembled. The invention embodies a virus which can express a fusion protein that consists of a viral protein and a fluorescent protein. In particular, the claimed invention relates to a virus that expresses ICP8-GFP.
The invention also includes methods for determining whether a cell is virus resistant. Such a method includes infecting the cell to be tested with a virus that expresses the fusion protein, and then detecting the presence or absence of the fusion protein. A fusion protein comprising a fluorescent protein can be identified by detecting the amount of fluorescence emitted by the protein. The invention also pertains to a method for identifying an anti-viral agent or an agent that blocks infection or other viral function and, thus, the expression of the fusion protein. Such a method can involve infecting a host cell with a virus as described herein and subjecting a host cell to the agent to be tested, and then detecting the presence of the fusion protein. A decrease in the amount of fusion protein indicates that the agent can be an anti-viral agent or an agent that blocks the expression of the fusion protein. Again, when the fusion protein contains a fluorescent protein, the amount of virus present can be detected by the amount of fluorescence emitted by the fusion protein. Another way of detecting the amount of herpesviral replication present can be by determining the amount of replication compartment formation.
The invention also embodies identifying an agent that reduces infection of a virus in vivo. Such a method includes infecting a mammal with a virus that expresses the fusion protein and then subjecting the mammal with the agent to be tested. One then removes a portion of the infected tissue and detects the amount of fusion protein that is produced. A decrease in the amount of fusion protein indicates that the agent is effective in treating the virus. Similarly, the invention can be used to assay for virus resistant cells.
The claimed invention specifically embodies a fusion protein having a herpesviral protein (e.g., ICP8), and the fluorescent protein (e.g., a green fluorescent protein (GFP)). The ICP8-green fluorescent protein is embodied by the claimed invention and its amino acid sequence is referred to herein as SEQ ID NO: 2. The claimed invention also relates to the nucleic acid sequence that encodes the ICP8-GFP fusion protein and is referred to herein as SEQ ID NO: 1.
The invention also relates to a kit that comprises a virus which is capable of expressing the fusion protein, as described herein. The kit can further comprise a complementing cell line or one that expresses the corresponding wild-type viral protein (e.g., S-2) and/or a cell line into which the virus can be transfected (e.g., Vero cell).
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Knipe David M
McNamee Elizabeth E.
Taylor Travis J.
Hamilton Brook Smith & Reynolds P.C.
Mosher Mary E.
President and Fellows of Harvard College
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