Protein targeting into HIV virions based on HIV-1 VPR fusion...

Details Protein targeting into HIV virions based on HIV-1 VPR fusion... Protein targeting into HIV virions based on HIV-1 VPR fusion...

1999-01-19

2002-10-22

Scheiner, Laurie (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Fusion protein or fusion polypeptide

C435S069700, C530S300000, C530S350000, C424S188100, C424S208100

Reexamination Certificate

active

06468539

ABSTRACT:

BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to two different approaches using the Vpr/Vpx protein or p6 protein for treatment of HIV-1 and HIV-2 infections.
(b) Description of Prior Art
Acquired Immune Deficiency Syndrome (AIDS) is a slow degenerative disease of the immune and nervous systems caused by the Human Immunodeficiency Virus (HIV). The life cycle of HIV lies at the heart of the AIDS pandemic. The spread of the disease is primarily determined by the infectious properties of this virus. Progressive lethal degeneration of the immune and central nervous systems results from long term chronic replication of this virus.
HIV belongs to a unique virus family, the retroviridae, a group of small, enveloped, positive stranded, RNA viruses (Lavallée et al., 1994, J. Virol, 68:1926-1934; International Patent Application No. WO 90/158,75 on Dec. 27, 1990 in the name of DANA FARBER CANCER INSTITUTE). These viruses code for an enzyme, the reverse transcriptase, which enables them to replicate their RNA genome through a DNA intermediate. Simple retroviruses Contain three, contiguous reading frames coding for the gag, pol and env genes, which constitute their structural and enzymatic repertoire, all packaged in the progeny virion. The gag and env genes encode the core nucleocapsids proteins and the membrane glycoproteins of the virus, respectively, whereas the pol gene gives rise to the reverse transcriptase and other enzymatic activities (ribonuclease H, integrase and protease) that are essential for viral replication. HIV belongs to the lentivirus subfamily, members of which are characterized by several additional open reading frames (ORF) not found in simple retroviruses (FIG.
1
). These ORFs all appear following gag-pol sequences, either immediately preceding the env sequences or overlapping it, and at least in one case, nef, extending well into the 3′ Long Terminal Repeat (LTR). These ORFs code for non-structural viral proteins readily detectable in the cells. Much evidence has accumulated indicating that these gene products, collectively referred to as auxiliary proteins , are capable of modulating viral replication and infectivity.
HIV-1 possesses at least six such auxiliary proteins, namely, Vif, Vpr, Tat, Rev, Vpu and Nef. The closely related HIV-2 does not code for Vpu, but codes for another unrelated protein, Vpx, not found in HIV-1. Mutations affecting either Tat or Rev severely impair viral replication indicating that these two auxiliary proteins are essential for viral replication. However, at least in vitro, mutations affecting other auxiliary proteins result in minimal effect on the viral replication kinetics. Hence, these proteins have been dubbed dispensable or non-essential for in vitro replication, and are usually referred to as accessory gene products.
In the past few years, it has become evident that while these “accessory” genes are not required for productive replication, they are nonetheless capable of affecting replication events, even in vitro. More importantly, recent data indicates that they may affect pathogenesis in vivo.
The vpr gene encodes a 14 kDa protein (96 amino acids) in most strains of HIV-1 (
FIG. 2
; Myers et al., 1993
, Human Retroviruses and AIDS
1993 I-II, Los Alamos National Laboratory, New Mexico, USA), although the open reading frame is often truncated in viral strains extensively passaged in tissue culture. The vpr open reading frame is also present in HIV-2 isolates and in most but not in all HIV isolates. A sequence similar to HIV-1 vpr is also found in Visna virus. Recent amino acid comparison between HIV-2 vpr and vpx showed regions of similarity suggesting that vpx in the HIV-2/SIV group may have arisen by duplication of the vpr gene (
FIG. 2
; Myers et al., 1993, Human Retroviruses and AIDS 1993 I-II, Los Alamos National Laboratory, New Mexico, USA). The Vpr protein is made from a singly spliced rev-dependent mRNA species that accumulates late in infection. The Vpr protein of HIV and SIV have recently been shown to be present in mature viral particles in multiple copies. Interestingly, Vpr and Vpx are the first regulatory protein of any retrovirus found to be associated with viral particles. Other regulatory proteins, such as tat, Rev, Nef, Vif and Vpu are not virion-associated. The assembly and maturation of HIV-1 viral particles is a complex process in which the structural Gag, Pol and Env gene products are expressed in the form of polyprotein precursors. The Gag proteins of HIV play a central role in virion assembly and budding. Gag proteins are initially synthesized as myristylated polyprotein precursors, Pr55
gag
and Pr160
gag-pol
, which are transported to the inner face of the plasma membrane where they can direct particle formation, even in the absence of other viral proteins. Complete budding leads to formation of immature particles, followed by HIV protease mediated cleavage of the Gag and Gag-Pol precursor polyproteins and formation of mature HIV particles with condensed core. The mature virion proteins derived from cleavage of the gag-encoded precursor, Pr55
gag
, include the p17 matrix protein (MA), the p24 capsid protein (CA), the p7 nucleocapsid protein (NC), and a small proline-rich peptide of approximately 6 kDa designated p6 which are linked in this order in the polyprotein precursor. Vpr is not part of the virus polyprotein precursors and its incorporation occurs by way of an interaction with a component normally found in the viral particle. It was recently reported that the HIV-1 Vpr could be incorporated in trans into viral-like particle (VLP) originating from expression of the Pr55
gag
only (Lavallée et al., 1994, J. Virol., 1926-1934). Data from this and other studies indicate that Vpr incorporation appeared to result from a direct interaction of Vpr with the carboxy-terminal region of the Pr55
gag
polyprotein (Paxton et al. 1993, Journal of Virology, 67(12):7229-7237; Lu et al., 1993, Journal of Virology, 67(1):6542-6550).
Functional studies indicated that the full length vpr protein could confer favorable growth properties to viruses. The increase in virion production is more pronounced in primary macrophages in both HIV-1 and HIV-2 systems, suggesting that Vpr function may be important in specific target cells. Interestingly, while mutations affecting HIV-1 vpr do not affect replication in peripheral blood mononuclear cells (PBMC), mutations in HIV-2 vpr results in a measurable impairment in these cells. Similarly, a recent study using anti-sense RNA directed against vpr inhibited viral replication in primary macrophages but not in transformed T-cells. Previous work indicated that this rapid growth advantage may be conferred by the weak transactivation property of Vpr on HIV-LTR directed gene expression (European Patent Application published under No. 474,797 on Mar. 18, 1992 in the name of DANA FARBER CANCER INSTITUTE). Cotransfection experiments suggest that vpr could augment the expression of a reporter gene from several heterologous promoters by approximately three to ten fold.
The carboxyl terminal sequence of Vpr have been shown to be important for Vpr mediated transactivation as prematurely truncated proteins are non-functional and are not packaged into the virion. Interestingly, a recent report also indicated that the carboxyl terminal of the protein is important for nuclear localization (Lu et al., 1993, Journal of Virology, 67(1):6542-6550). A specific vpr responsive LTR sequence was not identified and the exact mechanism by which vpr augments reporter gene expression is not clear. The precise mode of action of vpr is yet to be established. However, the presence of vpr in the viral particle suggests that this protein has a role in the early stage of infection. Virion-associated non-structural proteins in many viral systems play a pivotal enzymatic functions in early replication steps, either because cellular homologues are unavailable or are sequestered, for example, in the nucleus. It is possible that Vpr is one such protein, capable of modulating ea

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