Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Selective anticancer strategies via intervention of the death pathways relevant to cell transformation.

Apoptosis is an important physiological process that promotes tissue homeostasis by eliminating unnecessary or malfunctioning cells. Abnormality in this process contributes to tumorigenesis, as well as the resistance to cancer treatment by radiation and chemotherapy. Restoration of normal apoptosis would not only promote cancer cell death and halt tumor progression, but also increase the response to many current cancer therapies. Although apoptosis induction is an important principle of currently used radiation and chemotherapy treatment, uncovering the mechanisms that govern this process, and which are lost during transformation, represents an important direction for realizing improved therapies for the future. This article first briefly reviews aspects of current discovery strategies for new anticancer therapeutics based on intervening in cell death pathways, and then discusses in more detail several cancer-relevant death pathways, which are disabled during transformation and which can be targeted therapeutically. These include anoikis/cell adhesion; energy metabolism and the unfolded protein response. Finally, we introduce a new concept, which utilizes cancer-specific apoptosis induced by oncolytic viruses. The discussion of these topics involves novel targets, compounds and virotherapy.

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin.

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.

Site-directed mutagenesis of two trans-regulatory genes (tat-III,trs) of HIV-1.

Point mutations were introduced into the overlapping trans-regulatory genes (tat-III and trs) of human immunodeficiency virus type 1 (HIV-1), and the mutants were evaluated for virus expression. The results showed that tat-III has a positive transacting role and is required for transcriptional activation. A chain terminating mutation early in the trs gene resulted in an increase in transcription of viral messenger RNA as measured by nuclear transcription experiments, but only one major species of viral messenger RNA (1.8 kilobases) was detected, and little or no viral structural proteins were made. Thus, the trs gene product is essential for expression of virus structural proteins but, at the same time, may have a negative trans-regulatory role in transcription. Cotransfection of the point mutant proviruses defective in tat or trs with each other or with a complementary DNA clone containing tat and trs sequences restored the normal transcription pattern and subsequent virus production.

T-cell growth factor gene: lack of expression in human T-cell leukemia-lymphoma virus-infected cells.

Activated mature T cells require T-cell growth factor (TCGF) for continuous proliferation. However, many mature T cells infected with human T-cell leukemia-lymphoma virus grow independently of exogenously added TCGF. It is now reported that cells infected with this virus also lack detectable TCGF messenger RNA (less than one copy per cell) and thus do not produce their own growth factor. The results apparently rule out an autostimulation mechanism of growth control.

Human-proto-oncogene nucleotide sequences corresponding to the transforming region of simian sarcoma virus.

The nucleotide sequences of the six regions within the normal human cellular locus (c-sis) that correspond to the entire transforming region of the simian sarcoma virus (SSV) genome (v-sis) were determined. The regions are bounded by acceptor and donor splice sites and, except for region 6, resemble exons. Region 6 lacks a 3' donor splice site and terminates -5 base pairs from the 3' v-sis-helper-viral junction. This is consistent with a model proposing that SSV was generated by recombination between proviral DNA of a simian sarcoma associated virus and proto-sis and that introns were spliced out subsequently from a fused viral-sis messenger RNA. This also suggests that the 3' recombination occurred within an exon of the woolly monkey (Lagothrix) genome. The open reading frames predicting the v-sis and c-sis gene products coincide with the stop codon of c-sis located 123 nucleotides into the fifth region of homology. The overall nucleotide homology was 91 percent with substitutions mainly in the third codon positions within the open reading frame and with greatest divergence within the untranslated 3' portion of the sequences. The predicted protein products for v-sis and c-sis are 93 percent homologous. The predicted c-sis gene product is identical in 31 of 31 amino acids to one of the published sequences of platelet-derived growth factor. Thus, c-sis encodes one chain of human platelet-derived growth factor.

A cellular cofactor for the constitutive transport element of type D retrovirus.

A human nuclear protein that specifically interacts with the constitutive transport element (CTE) of simian retrovirus was identified as adenosine 5'-triphosphate-dependent RNA helicase A. This protein could bind to functional CTE but not to inactive CTE mutants. The interaction of helicase A with CTE was distinct from previously described helicase activity of this protein. Helicase A shuttled from the nucleus to the cytoplasm in the presence of a transcription inhibitor or in cells transiently overexpressing CTE-containing RNA. In vivo colocalization of helicase A and CTE was observed in experiments that combined in situ hybridization and immunostaining. These results suggest that helicase A plays a role in the nuclear export of CTE-containing RNA.

Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells.

The first step in the infection of human T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is attachment to the target cell receptor, the CD4 antigen. This step may be vulnerable to attack by antibodies, chemicals, or small peptides. Dextran sulfate (molecular weight approximately 8000), which has been given to patients as an anticoagulant or antilipemic agent for more than two decades, was found to block the binding of virions to various target T lymphocytes, inhibit syncytia formation, and exert a potent inhibitory effect against HIV-1 in vitro at concentrations that may be clinically attainable in human beings. This drug also suppressed the replication of HIV-2 in vitro. These observations could have theoretical and clinical implications in the strategy to develop drugs against HIV types 1 and 2.

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses.

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.

The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats.

Expression of the pX protein of human T-cell leukemia virus type I (HTLV-I) in animal cells demonstrates that this protein is a specific transcriptional activator of the long terminal repeats (LTR) of HTLV-I. Several other promoters are not affected by pX. No lymphocyte-specific factors are required for this activation. pX can be detected in the nucleus of transfected monkey kidney cells (line CV1) by indirect immunofluorescence. These results indicate that the pX protein is essential for the replication cycle of the virus and that it may be directly involved in the immortalization of human lymphocytes by HTLV-I.

Trans-activator gene of human T-lymphotropic virus type III (HTLV-III).

Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.

Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III.

The retrovirus involved in acquired immune deficiency syndrome (HTLV-III/LAV) contains a region that is necessary for stimulation of gene expression directed by the viral long terminal repeat. This region is located between nucleotides 5365 and 5607, immediately 5' to the envelope gene. A doubly-spliced message containing this region could encode an 86-amino acid protein with structural features similar to those of nucleic acid-binding proteins.

Structure of 3' terminal region of type II human T lymphotropic virus: evidence for new coding region.

The sequence of the 3' terminus of the human T lymphotropic virus type II (HTLV-II) was determined and compared to the corresponding sequence of HTLV-I. The 1557-nucleotide-long sequence can be divided into a 5' region that is not conserved between the two viruses, and a 3', 1011-nucleotide-long region that is highly conserved and that corresponds precisely with a long open reading frame for both HTLV-I and -II. The proteins that could be encoded by these open reading frames have a molecular weight of about 38,000 and are closely related in primary amino acid sequence. The genomic structure in the 3' region of HTLV was found to be similar to that of bovine leukemia virus.

The sor gene of HIV-1 is required for efficient virus transmission in vitro.

The genome of the human immunodeficiency virus HIV-1 contains at least eight genes, of which three (sor, R, and 3' orf) have no known function. In this study, the role of the sor gene was examined by constructing a series of proviral genomes of HIV-1 that either lacked the coding sequences for sor or contained point mutations in sor. Analysis of four such mutants revealed that although each clone could generate morphologically normal virus particles upon transfection, the mutant viruses were limited in their capacity to establish stable infection. Virus derived from transfection of Cos-1 cells (OKT4-) with sor mutant proviral DNA's was resistant to transmission to OKT4+ "susceptible" cells under cell-free conditions, and was transmitted poorly by coculture. In contrast, virus derived from clones with an intact sor frame was readily propagated by either approach. Normal amounts of gag-, env-, and pol-derived proteins were produced by all four mutants and assays in both lymphoid and nonlymphoid cells indicated that their trans-activating capacity was intact and comparable with wild type. Thus the sor gene, although not absolutely required in HIV virion formation, influences virus transmission in vitro and is crucial in the efficient generation of infectious virus. The data also suggest that sor influences virus replication at a novel, post-translational stage and that its action is independent of the regulatory genes tat and trs.

Identification of the fusion peptide of primate immunodeficiency viruses.

Membrane fusion induced by the envelope glycoproteins of human and simian immunodeficiency viruses (HIV and SIVmac) is a necessary step for the infection of CD4 cells and for the formation of syncytia after infection. Identification of the region in these molecules that mediates the fusion events is important for understanding and possibly interfering with HIV/SIVmac infection and pathogenesis. Amino acid substitutions were made in the 15 NH2-terminal residues of the SIVmac gp32 transmembrane glycoprotein, and the mutants were expressed in recombinant vaccinia viruses, which were then used to infect CD4-expressing T cell lines. Mutations that increased the overall hydrophobicity of the gp32 NH2-terminus increased the ability of the viral envelope to induce syncytia formation, whereas introduction of polar or charged amino acids in the same region abolished the fusogenic function of the viral envelope. Hydrophobicity in the NH2-terminal region of gp32 may therefore be an important correlate of viral virulence in vivo and could perhaps be exploited to generate a more effective animal model for the study of acquired immunodeficiency syndrome.

Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4+ T cells.

Studies of the biology and pathogenesis of Kaposi's sarcoma (KS) have been hampered by the inability to maintain long-term cultures of KS cells in vitro. In this study AIDS-KS-derived cells with characteristic spindle-like morphology were cultured with a growth factor (or factors) released by CD4+ T lymphocytes infected with human T-lymphotropic virus type I or II (HTLV-I or HTLV-II) or with human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2). Medium conditioned by HTLV-II-infected, transformed lines of T cells (HTLV-II CM) contained large amounts of this growth activity and also supported the temporary growth of normal vascular endothelial cells, but not fibroblasts. Interleukin-1 and tumor necrosis factor-alpha stimulated the growth of the KS-derived cells, but the growth was only transient and these could be distinguished from that in HTLV-II CM. Other known endothelial cell growth promoting factors, such as acidic and basic fibroblast growth factors and epidermal growth factor, did not support the long-term growth of the AIDS-KS cells. The factor released by CD4+ T cells infected with human retroviruses should prove useful in studies of the pathogenesis of KS.

Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat.

Human T-cell leukemia virus type III (HTLV-III) was recently identified as the probable etiologic agent of the acquired immune deficiency syndrome (AIDS). Here it is shown that, in human T-cell lines infected with HTLV-III, gene expression directed by the long terminal repeat sequence of this virus is stimulated by more than two orders of magnitude compared to matched uninfected cells. The rate of transcription of the HTLV-III long terminal repeat is more than 1000 times that of the SV40 early promoter in one infected cell line. Thus, HTLV-III, like HTLV-I, HTLV-II, and the bovine leukemia virus, is characterized by trans-activation of transcription in infected cells. The efficiency of trans-activation in the case of HTLV-III may account, at least in part, for the virulent nature of HTLV-III infection.

Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus.

A study was conducted of the genetic relation between human T-cell lymphotropic retroviruses and visna virus. The human T-cell lymphotropic viruses include those associated with T-cell malignancies (HTLV-I and HTLV-II) as well as the etiologic agent of the acquired immune deficiency syndrome (HTLV-III). Visna virus, a slowly replicating and pathogenic but nononcogenic retrovirus of sheep, is a member of the subfamily Lentivirinae. Results obtained by molecular hybridization and heteroduplex analysis indicated that a greater extent of nucleotide sequence homology exists between HTLV-III and visna virus than between HTLV-III and any of the other viruses. The homology observed under conditions of low stringency spanned the entire genome, but was strongest in the gag/pol region. The morphogenesis and fine structure of HTLV-III and visna virus also demonstrated striking similarities. The data provide strong evidence for a close taxonomic and thus evolutionary relation between HTLV-III and the Lentivirinae subfamily.

Characterization of long terminal repeat sequences of HTLV-III.

The nucleotide sequence of the long terminal repeat sequence (LTR) of the human T-cell leukemia (lymphotropic) virus type III (HTLV-III) was determined. This virus is associated etiologically with the acquired immune deficiency syndrome. The LTR was found to be 634 base pairs in length with U3, R, and U5 regions of 453, 98, and 83 bp, respectively. The proviral DNA is flanked by a 7-base-pair direct repeat. The promoter and polyadenylation signals are situated 27 and 24 base pairs upstream from the respective transcriptional initiation and polyadenylation sites. The primer binding site is complementary to transfer RNA-lysine. The LTR of HTLV-III, like that of HTLV-I, showed a limited homology to enhancer-like sequences within two genes expressed specifically in T lymphocytes, T-cell growth factor, and gamma-interferon. Structural comparisons revealed that the LTR of HTLV-III is distantly related to those of HTLV-I, HTLV-II, and bovine leukemia virus.