Genomics and drug discovery.

Genomics, the systematic study of all the genes of an organism, offers a new and much-needed source of systematic productivity for the pharmaceutical industry. The isolation of the majority of human genes in their most useful form is leading to the creation of new drugs based on human proteins, antibodies, peptides, and genes. Human Genome Sciences, Inc, was the first company to use the systematic, genomics approach to discovering drugs, and we have placed 4 of these in clinical trials. Two are described: repifermin (keratinocyte growth factor-2, KGF-2) for wound healing and treatment of mucositis caused by cancer therapy, and B lymphocyte stimulator (BLyS) for stimulation of the immune system. An anti-BLyS antibody drug is in advanced preclinical development for treatment of autoimmune diseases.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein.

The human immunodeficiency virus type 1 (HIV-1)-encoded vpu product is a small class 1 integral membrane protein that is phosphorylated by the ubiquitous casein kinase II (CKII) in HIV-1-infected cells. The Vpu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation. In this study, we investigated the role of phosphorylation in the biological activity of Vpu. Our results show that phosphorylation of Vpu occurs on serine residues at positions 52 and 56 located in a highly conserved dodecapeptide sequence. Mutation of either Ser 56, or both Ser 52 and Ser 56 impaired the ability of Vpu to delay the rate of syncytium formation while retaining virion release activity at levels comparable to vpu+ proviruses. Flow cytometry analysis indicates that the relative amounts of envelope glycoprotein gp120 expressed at the surface of cells transfected with these vpu mutant proviruses was two- to threefold greater than that observed on cells transfected with a vpu+ provirus. This increased expression of gp120 at the cell surface may explain the more rapid onset of syncytium formation observed in cell transfected with vpu mutant proviruses. These results suggest that Vpu-facilitated virion release and delayed cytopathic effect are the consequence of two distinct functional activities of the protein.

Integrase mutants of human immunodeficiency virus type 1 with a specific defect in integration.

A previous genetic analysis of the human immunodeficiency virus type 1 integrase protein failed to identify single amino acid substitutions that only block the integration of viral DNA (C.-G. Shin, B. Taddeo, W.A. Haseltine, and C.M. Farnet, J. Virol. 68:1633-1642, 1994). Additional substitutions of amino acids that are highly conserved among retroviral integrases were constructed in human immunodeficiency virus type 1 and analyzed for their effects on viral protein synthesis and processing, virion morphology, and viral DNA synthesis and integration in an attempt to identify mutants with a specific defect in integration. Four single amino acid substitutions resulted in replication defective viruses. Conservative, single amino acid substitutions of the two invariant aspartic acid residues found in all retroviral integrases prevented the integration of viral DNA and had no detectable effect on the other stages in the viral replication cycle, indicating that these mutants exhibited a specific defect in integration. Mutations at two positions, S-81 and P-109, blocked the integration of viral DNA but also resulted in the production of viral particles that exhibited reduced reverse transcriptase activity, suggesting additional defects in viral replication. Substitution of the highly conserved amino acid T66 had no effect on viral replication in a CD4+ human T-cell line. This analysis extends the range of possible phenotypes that may be produced by single amino acid substitutions in conserved residues of the integrase protein.

Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of man's commonest hereditary diseases. Several studies have implicated a defect in DNA mismatch repair in the pathogenesis of this disease. In particular, hMSH2 and hMLH1 homologues of the bacterial DNA mismatch repair genes mutS and mutL, respectively, were shown to be mutated in a subset of HNPCC cases. Here we report the nucleotide sequence, chromosome localization and mutational analysis of hPMS1 and hPMS2, two additional homologues of the prokaryotic mutL gene. Both hPMS1 and hPMS2 were found to be mutated in the germline of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.

Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein.

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms an inner coat directly underneath the lipid envelope of the virion. The outer surface of the lipid envelope surrounding the capsid is coated by the viral Env glycoproteins. We report here that the HIV-1 capsid-Env glycoprotein association is very sensitive to minor alterations in the MA protein. The results indicate that most of the MA domain of the Gag precursor, except for its carboxy terminus, is essential for this association. Viral particles produced by proviruses with small missense or deletion mutations in the region coding for the amino-terminal 100 amino acids of the MA protein lacked both the surface glycoprotein gp120 and the transmembrane glycoprotein gp41, indicating a defect at the level of Env glycoprotein incorporation. Alterations at the carboxy terminus of the MA domain had no significant effect on the levels of particle-associated Env glycoprotein or on virus replication. The presence of HIV-1 MA protein sequences was sufficient for the stable association of HIV-1 Env glycoprotein with hybrid particles that contain the capsid (CA) and nucleocapsid (NC) proteins of visna virus. The association of HIV-1 Env glycoprotein with the hybrid particles was dependent upon the presence of the HIV-1 MA protein domain, as HIV-1 Env glycoprotein was not efficiently recruited into virus particles when coexpressed with authentic visna virus Gag proteins.

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.

Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody.

A single-chain antibody, derived from a human monoclonal antibody that recognizes the CD4 binding region of the human immunodeficiency virus type 1 (HIV-1) envelope protein, has been designed for intracellular expression in eukaryotic cells. The single-chain antibody is composed of an immunoglobulin heavy-chain leader sequence and heavy- and light-chain variable regions that are joined by an interchain linker. The antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic to the cells. The antibody binds to the envelope protein within the cell and inhibits processing of the envelope precursor and syncytia formation. The infectivity of the HIV-1 particles produced by cells that express the single-chain antibody is substantially reduced. These studies illustrate the feasibility of designing antibodies that bind and inactivate molecules intracellularly. Antibodies that act on target molecules within cells should provide a useful tool for research as well as for control of infectious and other diseases.

Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses.

The Vpu protein of human immunodeficiency virus type 1 facilitates the release of virus particles from the surface of infected cells. The ability of the Vpu protein to facilitate release of Gag proteins from retroviruses that lack a Vpu-like protein was examined. The results of these experiments show that Vpu significantly increases the release of the Gag proteins of human immunodeficiency virus type 2, visna virus, and Moloney murine leukemia virus from HeLa cells. The results indicate that Vpu-mediated enhancement of particle release requires neither amino-terminal myristoylation of the Gag precursor nor cleavage of the Gag precursor by the viral protease. The results raise the possibility that Vpu modifies a cellular pathway common to the release of all retroviruses from the cell surface.

Early molecular replication of human immunodeficiency virus type 1 in cultured-blood-derived T helper dendritic cells.

The rate and efficiency of key steps in the life cycle of the human immunodeficiency virus type 1 was examined in three primary cell types, T cells, monocytes, and T helper dendritic cells using the same quantity of virus involved and same cell number. The results show that viral DNA synthesis proceeds much more rapidly and efficiently in primary T helper dendritic cell populations than in primary T cell and monocyte populations. The increased rate of virus DNA synthesis is attributable either to an increase in the efficiency and the rate of uptake of the virus particles by the T helper dendritic cells, as compared with that in other cell types, or to an increased efficiency and rate of viral DNA synthesis in the T helper dendritic cells. In the subsequent phase of viral expression the appearance of spliced viral mRNA products also occur more rapidly in cultures of primary-blood-derived T helper dendritic cells than is the case in primary T cells and monocytes. The increased efficiency of the early steps of HIV-1 replication in primary-blood-derived T helper dendritic cells than in other blood-derived mononuclear cells raises the possibility that these cells play a central role in HIV-1 infection and pathogenesis.

Effect of nef alleles on replication of human immunodeficiency virus type 1.

The effect of multiple alleles of nef of the human immunodeficiency virus type 1 (HIV-1) on virus replication was examined. Nef alleles used include some derived from isolates of virus passaged in tissue culture as well as others obtained by direct cloning of viral DNA from tissues of infected patients. The effect of nef on virus replication was evaluated in the context of a derivative of the HXB2 provirus shown previously to require nef for rapid growth in CD4+ human T cell lines and in primary peripheral blood mononuclear cells. The results of the experiments show that in this genetic context all of the studied viruses carrying nef alleles that express stable Nef proteins replicate more rapidly than do their, otherwise isogenic, nef-defective counterparts. Two of the nef alleles derived from primary tissues produce unstable proteins. These studies demonstrate that naturally occurring nef alleles can increase the rate of virus replication in both primary peripheral blood mononuclear cells and in a CD4+ T cell line. The results also demonstrate that functional variation exists among naturally occurring nef alleles.