Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-05-21
2001-07-10
Budens, Robert D. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007200, C435S007240, C435S372300
Reexamination Certificate
active
06258527
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the infection of target cells by HIV-1, and more particularly to agents identified herein that mediate the entry of macrophage-trophic HIV into such target cells, and to the diagnostic and therapeutic uses to which such agents may be put.
BACKGROUND OF THE INVENTION
The human immunodeficiency viruses infect CD4
+
macrophages and T helper cells. Although HIV-1 entry requires cell surface expression of CD4, to which the viral envelope glycoproteins bind, several studies have suggested that it is not sufficient for fusion of the viral envelope to the cellular plasma membrane. Early studies have shown that while human cells expressing a transfected CD4 gene were permissive for virus entry, murine cells expressing human CD4 were not. These findings led to the suggestion that there is a species-specific cell surface cofactor required in addition to CD4 for HIV-1 entry. Subsequent studies have shown that strains of HIV-1 that had been adapted for growth in transformed T-cell lines (T-tropic strains) could not infect primary monocytes or macrophages; in contrast, primary viral strains were found to infect monocytes and macrophages, but not transformed T cell lines. This difference in tropism was found to be a consequence of specific sequence differences in the gp120 subunit of the envelope glycoprotein, suggesting that multiple cell type-specific cofactors may be required for entry in addition to CD4.
The nature of the cofactors required for HIV entry proved elusive until the recent identification by Feng et al. of fusin, a member of the seven transmembrane G-protein coupled receptor family. Fusin (CXCR-4) was shown to act as a co-receptor for T-tropic strains; however, it did not support infection of CD4
+
cells by macrophage-tropic viruses, which more closely resemble those that predominate in infected individuals throughout the course of the disease, particularly in the asymptomatic phase. In addition, these strains appear to be responsible for HIV- 1 transmission, both sexually and by transfer of infected blood. Rare individuals who are resistant to sexual transmission of HIV-1 have T-cells that are readily infected by T-tropic virus, but cannot be infected by macrophage-tropic virus, further supporting a role for macrophage-tropic virus in sexual transmission of HIV-1.
Cocchi et al. recently characterized inhibitors of HIV-1 replication present in supernatants of CD8
+
T cells as the &bgr;p-chemokines RANTES, MIP-1&agr; and MIP-1&bgr;. Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in ref. 14). The chemokines fall into two classes, C-X-C (&agr;) and C-C (&bgr;), depending on whether the first two cysteines are separated by a single amino acid or are adjacent. The &agr;-chemokines such as IL-8, NAP-2 and MGSA are chemotactic primarily for neutrophils, while &bgr;-chemokines such as RANTES, MIP-1&agr;, MIP-1&bgr;, MCP-1, MCP-2, and MCP-3 are chemotactic for macrophages, T-cells, eosinophils and basophils. The chemokines bind specific cell surface receptors belonging to the family of G protein-coupled seven transmembrane domain proteins (reviewed in Ref. 15). Upon binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated trimeric G protein. This results in a rapid increase in intracellular calcium concentration. There are at least seven human chemokine receptors that bind or respond to &bgr;-chemokines with the following characteristic pattern: CC-CKR1 (MIP-1&agr;, MIP-1&bgr;, MCP-3, RANTES), CC-CKR-2A and CC-CKR-2B (MCP-1, MCP-3), CC-CKR-3 (eotaxin, RANTES, MCP-3), CC-CKR-4 (MIP-1&agr;, RANTES, MCP-1), CC-CKR-5 (MIP-1&agr;, RANTES, MIP-1&bgr;), and the Duffy blood group antigen (RANTES, MCP-1). These transmembrane receptors could be involved in HIV infection.
Therefore, there is a need to identify a translocation promoting agent that functions in conjunction with CD4 during HIV infection in macrophage. Further, there is a need to provide methods for identifying drugs that can interfere with HIV infection of macrophage by hindering the interaction of CD4, the translocation promoting agent and HIV envelope proteins.
SUMMARY OF THE INVENTION
In its broadest aspect, the present invention relates to the identification and application of an agent capable of promoting the translocation of macrophage-trophic HIV through the membrane of a target CD4
+
cell, which agent exhibits certain of the following characteristics and activities:
A. It is present in, on, or proximal to the cell membrane of the target CD4
+
cell;
B. It acts in tandem with CD4, in connection with the translocation; and
C. It is capable of interacting with associated G-proteins to thereby transduce an intracellular signal.
A further characteristic attendant to the activity of the translocation promoting agent of the present invention is an observed increase in the concentration of intracellular calcium. The present agent may also be described as a mediator of the entry of envelope glycoproteins of macrophage-trophic strains of HIV-1 into target cells.
In a further aspect of the invention, the present translocation promoting agent appears to act in conjunction with CD4 in facilitating the penetration of the macrophage-trophic virus into the target cell to establish HIV infection. A particular family of receptors known as C-C (or &bgr;) chemokine receptors (CKRs) has been identified as defining certain of the activities and characteristics set forth above, and a specific such receptor, CC-CKR5, is exemplified herein.
Other analogous receptors, such as those encoded by some viruses, particularly members of the Herpes virus family (CMV, HHV-6, HHV-8), serve to broaden the host range of HIV in individuals infected with both HIV and these viruses. This may therefore increase the range of tissues infected or provide a ligand for HIV envelope that may result in deleterious signal transduction in various tissues. Such information could lead to novel approaches to block the synergy between HIV and viral cofactors.
The present invention also relates to the use of a recombinant DNA molecule or cloned gene, or a truncated or degenerate variant thereof, which encodes a translocation promoting agent or the active portion thereof; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene. In another embodiment, the human and murine DNA sequences of the translocation promoting agent of the present invention, or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the translocation promoting agent. For example, the probes may be prepared with a variety of known vectors, such as the phage &lgr; vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences so prepared or constituted. Correspondingly, the preparation of antisense RNA and ribozymes are included herein.
The present invention also includes translocation promoter agents having the activities noted herein. In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present translocation promoter agent(s).
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to pr
Deng Hongkui
Ellmeier Wilfried
Landau Nathaniel R.
Littman Dan R.
Liu Rong
Budens Robert D.
Klauber & Jackson
The Aaron Diamond Aids Research Center
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