A reporter system for replication-competent gammaretroviruses: the inGluc-MLV-DERSE assay.

Although novel retroviral vectors for use in gene-therapy products are reducing the potential for formation of replication-competent retrovirus (RCR), it remains crucial to screen products for RCR for both research and clinical purposes. For clinical-grade gammaretrovirus-based vectors, RCR screening is achieved by an extended S(+)L(-) or marker-rescue assay, whereas standard methods for replication-competent lentivirus detection are still in development. In this report, we describe a rapid and sensitive method for replication-competent gammaretrovirus detection. We used this assay to detect three members of the gammaretrovirus family and compared the sensitivity of our assay with well-established methods for retrovirus detection, including the extended S(+)L(-) assay. Results presented here demonstrate that this assay should be useful for gene-therapy product testing.

Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors.

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA.

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.

Ternary complex factors and cofactors are essential for human T-cell leukemia virus type 1 tax transactivation of the serum response element.

The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription.

Comparisons of defective HTLV-I proviruses predict the mode of origin and coding potential of internally deleted genomes.

Cell lines infected with a variety of HTLV-I isolates were examined for the presence of defective proviruses that contain deletions spanning the gag, pol, and env genes. Internally deleted proviruses were identified by Southern blotting and by PCR amplification with 5' and 3' primers complementary to gag and tax sequences, respectively. PCR products representing eight defective proviruses from seven different cell lines were subsequently cloned and sequenced. The objectives of this study were twofold: first, we sought to determine whether nucleotide sequences surrounding sites of deletion shared common features that might reveal the mechanisms by which the defective genomes originated. Second, we asked whether deleted proviruses encode Gag fusion proteins with related C-terminal residues derived from open reading frames in the pX region. While most of the defective proviruses had incurred a single, large deletion, two of them displayed a more complex pattern of multiple rearrangements. Alignments of bases flanking the 5' and 3' deletion endpoints within each provirus showed tracts of sequence identity consistent with a mechanism involving aberrant intramolecular strand-transfer events during replication. We suggest that the amount or activity of HTLV-I polymerase in virions may contribute both to the poor infectivity of the virus and to the high deletion frequency. Two of the eight proviruses that were examined encoded a gag gene joined to an extended open reading frame; the other six had very short open reading frames (one to six amino acids) derived from pX or env regions joined to gag that showed no apparent amino acid sequence similarity.

Defective and wild-type human T-cell leukemia virus type I proviruses: characterization of gene products and trans-interactions between proviruses.

Defective provirus genomes of human T-cell leukemia virus type I are frequently detected in lymphocytes from infected individuals and in infected cell lines. One type of defective provirus contains internal deletions spanning gag, pol, and env genes but retains portions of open reading frames for trans-regulatory proteins. The deleted proviruses could potentially contribute to viral pathology by producing novel gene products that directly affect cell metabolism or that modulate expression of resident, wild-type proviruses. Virus gene products and the control of their expression were examined in cells transfected with defined molecular clones of wild-type and defective proviruses. Internally deleted provirus clones, which are unable to produce functional Tax and Rex proteins, were transcriptionally inactive in transfected cells. Ectopic expression of p40Tax activated transcription of the deleted provirus, resulting in the accumulation of a two-exon mRNA that yields a truncated form of Rex (p21Rex). Although this two-exon mRNA also has a potential initiation codon in the tax frame, a truncated form of Tax was not detected by immunoblotting or in transactivation assays. When complemented with p40Tax and p27Rex, cells transfected with deleted proviruses accumulated an unspliced mRNA that could potentially encode gag-pX fusion proteins. Although expression of deleted proviruses was dependent on trans-acting factors produced from intact proviruses, gene products from defective proviruses did not significantly affect expression of a cotransfected, full-length provirus.

HIV type 1 protease inhibitors fail to inhibit HTLV-I Gag processing in infected cells.

Protease inhibitors are currently the most effective antiviral agents against human immunodeficiency virus type 1 (HIV-1). In this study we determined the effect of four HIV-1 protease inhibitors on human T cell leukemia virus type 1 (HTLV-I). Rhesus monkey cells infected with HTLV-I were treated with different concentrations of indinavir, saquinavir, ritonavir, or nelfinavir. The effect of these inhibitors was monitored through their effect on the processing efficiency of the viral Gag protein in cells, the natural substrate for the viral protease. These inhibitors failed to block processing of HTLV-I Gag. To confirm these findings, human cells were cotransfected with plasmids encoding infectious copies of HIV-1 and HTLV-I, and the cells were subsequently treated with these same HIV-1 protease inhibitors. At concentrations between 5 and 50 times the IC50 for inhibition of HIV-1 replication, inhibition of HIV-1 Gag cleavage was apparent. In contrast, no effect on HTLV-I Gag processing was seen. At higher concentrations, HIV-1 Gag processing was essentially completely inhibited whereas HTLV-I Gag cleavage was still unaffected. Thus, these inhibitors are not effective inhibitors of HTLV-I Gag processing. Sequence alignments of the HIV-1 and HTLV-I viral proteases and processing sites suggest that the active site of the HTLV-I protease may have subtle differences in substrate recognition compared with the HIV-1 protease.

Differential requirements for alternative splicing and nuclear export functions of equine infectious anemia virus Rev protein.

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.

Inhibition of acute-, latent-, and chronic-phase human immunodeficiency virus type 1 (HIV-1) replication by a bistriazoloacridone analog that selectively inhibits HIV-1 transcription.

Nanomolar concentrations of temacrazine (1,4-bis[3-(6-oxo-6H-v-triazolo[4,5,1-de]acridin-5-yl)amino-propyl ]piperazine) were discovered to inhibit acute human immunodeficiency virus type 1 (HIV-1) infections and suppress the production of virus from chronically and latently infected cells containing integrated proviral DNA. This bistriazoloacridone derivative exerted its mechanism of antiviral action through selective inhibition of HIV-1 transcription during the postintegrative phase of virus replication. Mechanistic studies revealed that temacrazine blocked HIV-1 RNA formation without interference with the transcription of cellular genes or with events associated with the HIV-1 Tat and Rev regulatory proteins. Although temacrazine inhibited the in vitro 3' processing and strand transfer activities of HIV-1 integrase, with a 50% inhibitory concentration of approximately 50 nM, no evidence of an inhibitory effect on the intracellular integration of proviral DNA into the cellular genome during the early phase of infection could be detected. Furthermore, temacrazine did not interfere with virus attachment or fusion to host cells or the enzymatic activities of HIV-1 reverse transcriptase or protease, and the compound was not directly virucidal. Demonstration of in vivo anti-HIV-1 activity by temacrazine identifies bistriazoloacridones as a new class of pharmaceuticals that selectively blocks HIV-1 transcription.

X-I and X-II open reading frames of HTLV-I are not required for virus replication or for immortalization of primary T-cells in vitro.

In contrast to other retroviruses of the oncovirinae subgroup, the primate and bovine leukemia viruses (HTLV, STLV, and BLV) encode genes in the X-region of the genome, between the env gene and the 3' long terminal repeat. In HTLV-I, two overlapping open reading frames (ORFs) in the distal half of the X-region encode tax and rex genes, while two ORFs (X-I and X-II) in the proximal half of this region potentially encode proteins designated p12(XI) (or rof) and p30(XII) (or tof). The biological functions and mechanisms of tax and rex have been studied extensively whereas the roles of the other ORFs have not yet been established. To identify possible functions for ORFs X-I and X-II, an infectious molecular clone of HTLV-I and a mutant provirus lacking these ORFs were compared with respect to virus replication, gene expression, and ability to immortalize primary T-cells. When transiently transfected into 293 cells, both intact and deleted proviruses directed the synthesis of virus mRNAs and proteins that were quantitatively and qualitatively identical. These viruses were also indistinguishable in their abilities to infect and replicate in DBS-FRhL cells, which are permissive for HTLV-I propagation. Immortalized T-cell lines were established after cell-free or coculture methods for infection of activated, human peripheral blood or cord blood lymphocytes with each of the cloned viruses. The growth kinetics, cytokine dependence, and cell surface markers of the infected T-cell cultures were similar for each provirus clone. Thus, ORFs X-I and X-II are not essential for virus infectivity, replication, gene expression, or T-cell immortalization in vitro.

Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products.

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Examining the molecular genetics of HTLV-I with an infectious molecular clone of the virus and permissive cell culture systems.

Infectious molecular clones of HTLV-I proviruses have only recently been reported. The long wait for such provirus clones reflects the difficulties inherent in propagating HTLV-I in vitro, and thus a rigorous demonstration of infectivity has awaited improved cell culture systems and sensitive detection techniques for HTLV-I. An intact HTLV-I provirus, originating from an American ATL patient, was subcloned into a plasmid vector and was designated pCS-HTLV. Transient transfections of mammalian cells with pCS-HTLV resulted in the synthesis of viral proteins and mRNAs which were assembled into virions that had physical and morphological characteristics typical of HTLV-I particles. The ability of these virus particles to infect cells, replicate, and produce infectious progeny was demonstrated initially in short term, cell-free infection assays by monitoring the expression of specific viral mRNAs. These studies have been extended in cell culture systems that support continuous virus production. Primary T-lymphocytes have been infected either with cell-free supernatant fluids from, or by coculture with, cells transiently transfected with pCS-HTLV, giving rise to continuous, IL-2-dependent cell lines that have been in culture for >1 year. Furthermore, fetal rhesus lung cells (FRhL) were shown to be permissive for HTLV-I replication and sustained virus expression after infection with pCS-HTLV. Continuous FRhL cell lines now have been established that express various HTLV-I proviruses and mutants. These provirus clones and cell lines provide us with the means to address long-standing questions dealing with the biology of HTLV-I.

Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells.

The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.

Detection of human T-cell leukaemia virus 1 permissive cells using cell lines producing selectable recombinant virions.

A selectable retrovirus vector based on a full length HTLV-1 provirus clone, pCS-HTLV-1, was constructed by replacing the coding regions for tax, rex and the 3' region of env with the prokaryotic neomycin resistance gene under the control of the CMV promoter. This vector, pHTLV-1-CMVneo, was transfected into HTLV-1 infected human lymphocytes and fibroblasts. The production of recombinant virus by these cells was measured by the transfer of G418 resistance to target cells. Infection of target cells showed a preference for human lymphocytes in addition to two human fibroblast cell lines, Hos7 and RD4, and the African green monkey kidney cell line, Cos7. This system provides a method to study the cellular tropism of HTLV-1 and additionally provides a model to facilitate molecular studies of the natural events of HTLV-1 infection and integration.

Equine infectious anemia virus trans-regulatory protein Rev controls viral mRNA stability, accumulation, and alternative splicing.

The cis- and trans-acting components of the Rev regulatory pathway employed by equine infectious anemia virus (EIAV) to regulate and coordinate viral gene expression were examined in complementation experiments. Viral protein expression and mRNA expression were compared in cells transiently transfected with wild-type or mutant proviruses in combination with Rev expression plasmids. Mutation of the predicted rev gene abolished Gag protein synthesis, and this defect was complemented, in trans, by Rev. Analysis of viral mRNAs from transfected cells confirmed that EIAV expresses five major mRNAs: the full-length and singly spliced mRNAs contain introns and encode viral structural proteins while the three fully spliced mRNAs, encoding nonstructural genes, are generated by alternative splicing. Compared to cells transfected with the wild-type provirus, the intron-containing mRNAs produced from the rev-minus mutant were present at reduced levels in the nuclear RNA fraction and were not detected in the cytoplasm. This pattern of viral mRNA synthesis was restored to the wild-type pattern by providing Rev in trans. In contrast to the intron-containing mRNAs, cytoplasmic accumulation of the multiply spliced class of mRNAs was independent of Rev. Closer examination of the multiply spliced class of viral mRNAs by reverse transcriptase-PCR analysis revealed a Rev-dependent alternative splicing phenomenon. In the absence of Rev, proviruses expressed a four-exon mRNA at high levels; the addition of Rev caused both a decrease in the levels of the four-exon mRNA and the appearance of a related mRNA lacking exon 3. The cis-acting RNA elements that mediate Rev responsiveness were studied with deleted proviruses, which revealed that EIAV contains at least two elements located near the ends of envelope gene. Unlike the Rev-responsive elements in other retroviruses, the cis-acting regions of EIAV do not appear to form complex secondary structures.

Posttranscriptional effector domains in the Rev proteins of feline immunodeficiency virus and equine infectious anemia virus.

By systematically dissecting the Rev proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV), we have identified within each a short peptide that is functionally interchangeable with the effector domains found in Rev-like proteins from other retroviruses. The active sequences from FIV and EIAV differ in several respects from other known effectors and may represent a distinct class of effector domain.

Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system.

To develop a reporter system to study the response of an integrated retroviral LTR and cellular and viral events which influence transcription, the 5' LTR of HTLV-1 was coupled to the Escherichia coli beta-galactosidase gene (lacZ). This construct was assembled within a vector containing the neomycin resistance gene controlled by the SV40 promoter, and introduced into HeLa cells. Expression from the LTR in one clone was upregulated by positive regulators of HTLV-1 expression, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and the HTLV-1 transregulatory protein (tax), as has been previously reported using transient transfection assays. This method proved to be a rapid and reproducible assay for the measurement of integrated viral LTR activation in a single cell system.

Protein interactions with DNA elements in variant equine infectious anemia virus enhancers and their impact on transcriptional activity.

The long terminal repeats (LTRs) from various cloned equine infectious anemia virus (EIAV) proviruses differ significantly, but all contain cis-acting DNA elements identical to MDBP-, PEA2-, AP-1-, and PU.1 (ets)-binding sites. A prototype EIAV LTR would contain one of each of these conserved elements. The LTR variations originate from the insertion of novel sequences between the PEA2 and AP-1 elements in the transcriptional enhancer unit. Viewed in this way, the LTR from provirus clone lambda 12 has an 11-bp insertion containing a PEA2 site and the LTR of the lambda 6 provirus has a 31-bp insertion/duplication containing PEA2, AP-1, and PU.1 sites. Two other LTRs were cloned by amplification of cDNAs from the persistently infected cell line, EIAV-FEA. A third LTR was generated by site-directed mutagenesis of one of the LTRs from EIAV-FEA cells. The latter three had a single base change in the element next to the TATA box that abolished PU.1 binding; however, the variable regions of these LTRs were shown by gel mobility shift assays to contain one or two PU.1 sites. One variable region was shown to have an octamer site overlapping its tandem PU.1 elements. Basal, PMA-activated, and Tat trans-activated transcriptional activities of the LTRs were compared in several different cell lines by transient transfection. The various promoters displayed different relative levels of activity depending on the cell line used and the condition of activation. This natural set of variant promoters may help define how changes in the components of the transcription complex influence transactivation by Tat. The diverse LTRs could endow their respective proviruses with a unique pattern of expression and activation in vivo.