Internally controlled quantitative assays for PKR mRNA and protein from liver biopsies and other finite clinical samples.

A method to quantify double-stranded RNA-dependent protein kinase (PKR) mRNA and protein from human cells is described. A competitive RT-PCR assay has been developed by synthesis of an internal standard control (ISC) species of RNA. A competitive immunoblot assay was used to quantify full-length PKR (FL-PKR) protein in a sample of total cellular proteins, using truncated PKR protein as an internal standard against which FL-PKR protein could be quantified. The method can be used for simultaneous analysis of transcriptional and postranscriptional regulation of PKR gene expression from very small clinical specimens such as liver biopsies, e.g., 2-3 mg (wet weight) and containing only 2x10(5) cells. To the best of our knowledge, this is the first report of a sensitive simultaneous assay system for this important immunoeffector molecule.

The mechanism of trans-activation of the MDR1 gene by human T-cell leukemia virus.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. We have recently demonstrated that the MDR1 promoter is transcriptionally activated by the HTLV-I tax protein, providing an explanation for the development of drug resistance in HTLV-I infections. Here we report that HTLV-I mediated MDR1 activation is dependent on the presence of an NF-IL6-binding site located between base pairs -148 and -141 relative to the transcription start site. This finding opens up the possibility of moderating P-gp expression through interference with NF-IL6 binding to its trans recognition element and subsequent repression of MDR1 transcription.

Enhanced MDR1 gene expression in human T-cell leukemia virus-I-infected patients offers new prospects for therapy.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I-infected subjects was investigated. Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp-mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that the MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.

Production of non-infectious human immunodeficiency virus-like particles which package specifically viral RNA.

A recombinant vector that rapidly produces large amounts of human immunodeficiency virus (HIV) virus-like particles (VLPs) was constructed. This vector lacks LTR sequences and a functional nef gene. The VLPs produced are non-infectious but similar in structure to mature, infectious HIV virions. They package specifically HIV RNAs containing appropriate signals and do not package abundant cellular mRNAs (e.g. actin). In the system described here, efficient particle production and release is decoupled from infection. Use of this VLP system offers many advantages over the study of infectious virions, permitting the expression of mutant phenotypes which interfere with virus infectivity.

Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1.

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop. In the guanine tetrad model interstrand guanine contacts from the dimer. We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism.

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.

L-arginine: a therapeutic option for AIDS/HIV infection?

Numerous studies implicate cellular immunological effector systems in the partial containment of virus replication during the early stages of HIV infection. Immunostimulatory therapeutic regimes designed to enhance virus clearance are therefore theoretically attractive, but are accompanied by the risk of concomitant activation of HIV replication. Supra-normal levels of L-arginine have been shown to induce broad immune stimulation in vitro and in vivo, but do not increase HIV gene expression in vitro. These observations, together with the lack of toxicity of this agent, suggest a novel therapeutic approach to HIV disease.

Molecular determination of cell tropism of human immunodeficiency virus.

In the 10 years since AIDS was first identified, knowledge of the causative agent, human immunodeficiency virus (HIV), has advanced remarkably. Molecular biological analysis has had a greater impact on the investigation of HIV and AIDS than on that of any other disease. Unfortunately, the vast amount of material published on this subject has still not resulted in a thorough understanding of the pathogenesis of AIDS. Undoubtedly, part of the reason for the complexity of this disease stems from the ability of HIV to infect a large number of different cell types. To comprehend the mechanisms involved in the pathogenesis of AIDS, it is of great importance to understand the factors that control the cell tropism of the virus. In this review, we describe the known factors involved in the determination of the cell tropism of retroviruses and the ways by which molecular biological analysis of HIV has revealed the nature of the processes involved in control of the tropism of the virus for various cell types.

The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism.

Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates.

Human immunodeficiency virus type 1 T-cell tropism is determined by events prior to provirus formation.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary in the ability to replicate in cells that bear the HIV-1 receptor, CD4. The mechanism responsible for these cell tropism differences is unknown. We examined different isolates of HIV-1 with regard to replication in specific tumor-derived CD4-positive T-cell lines and normal peripheral blood lymphocytes. To investigate early events in the virus life cycle at low multiplicities of infection, we used a modification of the polymerase chain reaction method. Use of a molecularly cloned primary HIV-1 isolate, HIV-1 JR-CSF, restricted for replication in T-cell lines, demonstrated that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. However, transfection of proviral DNA resulted in efficient transient virus production from these cells. Therefore, we conclude that at least one block to infection for HIV-1 strains in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.

Studies of heterologous promoter trans-activation by the HTLV-II tax protein.

The tax protein of HTLV-II increases the level of steady-state mRNA produced from the HTLV-II long terminal repeat (LTR) and also activates heterologous promoters. We have previously shown that the adenovirus E3 promoter, which is trans-activated by the adenovirus E1a protein, is also trans-activated by the tax protein. To investigate the mechanism of trans-activation by tax, we analyzed E3 promoter deletion mutants to determine nucleotide sequence requirements for activation of this promoter. Our results show that removal of different upstream regions within the promoter does not result in loss of trans-activation, indicating that tax does not appear to interact with a single DNA binding protein to activate the E3 promoter. In addition, tax and E1a together activate the E3 promoter in a greater than additive fashion, suggesting that these proteins function differently. Possible mechanisms of activation by the tax protein are discussed.

In vitro mutagenesis of the human T-cell leukemia virus types I and II tax genes.

The tax gene of the human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) is essential for viral replication and acts by increasing the level of RNA transcription. The tax genes of HTLV-I and HTLV-II encode proteins of 40 and 37 kilodaltons, respectively. By in vitro mutagenesis of the tax gene, we have investigated those regions of the protein which are essential for its function. Mutation of either the amino- or carboxy-terminal domain of the protein resulted in loss of trans-activation ability. In addition, specificity of its activity with regard to trans-activation of either the HTLV-I or HTLV-II long terminal repeats was conferred by the first 59 amino acids.

HTLV-II transactivation is regulated by the overlapping tax/rex nonstructural genes.

The human T-cell leukemia virus (HTLV) types I and II have two nonstructural genes that are encoded in overlapping reading frames. One of these genes, known as tax, has been shown to encode a protein responsible for enhanced transcription (transactivation) from the viral long terminal repeats (LTRs). Genetic evidence indicates that the second nonstructural gene of HTLV-II, here designated rex, acts in trans to modulate tax gene-mediated transactivation in a concentration-dependent fashion. The rex gene may regulate the process of transactivation during the viral life cycle.

HIV-1 production from infected peripheral blood T cells after HTLV-I induced mitogenic stimulation.

The human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are two distinct human retroviruses that infect T cells. Recent epidemiologic studies have identified a cohort of individuals that are coinfected with both viruses. It is reported here that human peripheral blood leukocytes infected with HIV-1 in vitro can be induced to produce large quantities of HIV-1 after mitogenic stimulation by noninfectious HTLV-I virions. It is also shown that HTLV-I virions may exert this effect prior to, immediately following, or well after the cells are infected with HIV-1. These results provide further impetus for epidemiologic studies of dually infected individuals to determine whether HTLV-I may act as a cofactor for acquired immunodeficiency syndrome (AIDS).

HTLV x gene mutants exhibit novel transcriptional regulatory phenotypes.

The human T-cell leukemia viruses, HTLV-I and HTLV-II, contain a gene, termed x, with transcriptional regulatory function. The properties of the x proteins were analyzed by constructing mutant genes containing site-directed deletions and point mutations. The results demonstrate that the amino terminal 17 amino acids of the x protein constitute part of a functional domain that is critical for the transcriptional activating properties of the protein. Within this region, substitution of a leucine residue for a proline residue results in major changes in the trans-activation phenotype of the protein. The mutant HTLV-II x protein, though incapable of activating the HTLV-II long terminal repeat, will block trans-activation of the HTLV-II long terminal repeat by the wild-type protein. The altered phenotype of this mutant suggests a potential negative regulatory function of the x protein.

U3 sequences from HTLV-I and -II LTRs confer pX protein response to a murine leukemia virus LTR.

Human T-cell leukemia virus (HTLV) types I and II are unusual among replication-competent retroviruses in that they contain a fourth gene (chi) necessary for replication. The chi gene product, p chi, transcriptionally transactivates the viral long repeat (LTR), and is thus a positive regulator. To investigate p chi transactivation, sequences from the U3 regions of the LTRs of HTLV-I and -II were inserted into the Moloney murine leukemia virus (M-MuLV) LTR by recombinant DNA techniques. Transient expression assays of the chimeric LTRs indicated that the HTLV sequences conferred to the M-MuLV LTR responsiveness to HTLV p chi protein. M-MuLV enhancers were not required for function of the chimeric LTRs. Infectious recombinant M-MuLVs containing chimeric LTRs were also generated. These viruses showed higher infectivity when assayed in mouse cells expressing HTLV-II p chi protein compared to normal mouse cells. Thus the HTLV sequences were able to confer p chi responsiveness to infectious M-MuLV. The generation of a virus dependent on a transactivating protein for its replication has implications for the evolution of the human T-cell leukemia viruses.