An AUG codon upstream of rev and env open reading frames ensures optimal translation of the simian immunodeficiency virus Env protein.

The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.

Human papillomavirus infection and its association with HIV.

Human papillomavirus (HPV) can infect oral mucosa, causing asymptomatic infection or warty lesions. Several case-control studies have confirmed HPV as an independent risk factor for squamous cell carcinoma. HPV-related cancers seem to have better prognoses and different risk factors than do HPV-negative ones. HIV-infected patients are known to be at increased risk for persistent genital and anal high-risk HPV infections and intraepithelial neoplasm. Since the era of highly active antiretroviral therapy, the prevalence and persistence of warty lesions in oral mucosa have increased. Oral squamous cell carcinoma was recently added in the case definitions for common HIV-related oral mucosa lesions. The increased risk of HPV infection in HIV patients has been associated with impaired immune response to HPV, highly active antiretroviral therapy, aging of the HIV-infected patients, and direct interaction between the 2 viruses. HPV32 seems to be much more prevalent in asymptomatic HPV infections and warts among those infected with HIV than among those in the general population. Regarding HIV genes, there is evidence of an interaction between HPV and tat, rev, and vpr. HIV might play a role in HPV-associated pathogenesis by exhorting oncogenic stimuli via tat and rev or visa versa.

Structural basis of HIV-1 resistance to AZT by excision.

Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.

Safety and efficacy of a lentiviral vector containing three anti-HIV genes--CCR5 ribozyme, tat-rev siRNA, and TAR decoy--in SCID-hu mouse-derived T cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes--namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme--have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

OBJECTIVE: Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. METHODS: In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. RESULTS: Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. CONCLUSIONS: The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

Inhibition of multidrug-resistant HIV-1 by interference with cellular S-adenosylmethionine decarboxylase activity.

S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, can be specifically inhibited by the experimental drug SAM486A. The pharmaceutical interference with SAMDC activity results in the depletion of the intracellular pool of spermidine and spermine. In particular, low spermidine levels compromise hypusine modification and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), which is a cellular cofactor of the essential human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev. In the present study, we show that SAM486A efficiently suppresses HIV-1 replication, including the replication of viruses that are resistant to multiple reverse transcriptase and protease inhibitors. At drug concentrations that efficiently inhibit the formation of progeny viruses, no toxic effects of SAM486A on cellular metabolism are observed. It is demonstrated that the antiretroviral effect of SAM486A is based on the fact that Rev activity is severely compromised in drug-treated cells. Thus, inhibition of cellular SAMDC activity may provide a novel strategy to achieve suppression of otherwise drug-resistant viruses.

Potent inhibition of HIV-1 gene expression and TAT-mediated apoptosis in human T cells by novel mono- and multitarget anti-TAT/Rev/Env ribozymes and a general purpose RNA-cleaving DNA-enzyme.

One of the hallmarks of progression of HIV-1/AIDS is the rapid depletion of CD4+T cells that is known to occur at the late stages of the disease when usually X4 tropic HIV-1 predominates. Besides direct killing of T lymphocytes, HIV-1 infection leads to extensive apoptosis of naïve/uninfected bystander T cells, which is predominantly mediated by HIV-1 TAT protein. Therefore, reduction of HIV-1 TAT protein is likely to reduce substantially the pathogenesis associated with HIV-1 infection. We designed two non-GUX hammerhead ribozymes (Rzs) and a Di-Rz by placing them in direct tandem. These were targeted against the most conserved second exon of HIV-1 TAT/Rev/Env region. Although very impressive in vitro cleavage of the target RNA by the two hammerhead Rzs was obtained under standard conditions of cleavage, only one of them was active under simulated physiological conditions. Sequence-specific cleavage by the Di-Rz was most efficient under standard conditions. Cleavage reactions carried out under simulated physiological conditions by the Di-Rz, however, indicated that both mono-Rzs were functional. We also assembled a 10-23 catalytic motif containing general purpose RNA-cleaving DNA-enzyme (Dz) against the same region, which cleaved the target RNA very efficiently. Both Rzs and Dz showed not only potent inhibition of HIV-1 gene expression but also showed remarkable protection against HIV-1 TAT protein-mediated apoptosis in Jurkat T cells. Possible implications of these findings are discussed.

A single administration of lentiviral vectors expressing either full-length human immunodeficiency virus 1 (HIV-1)(HXB2) Rev/Env or codon-optimized HIV-1(JR-FL) gp120 generates durable immune responses in mice.

Genetic immunization using viral vectors provides an effective means to elicit antigen-specific cellular immune responses. Several viral vectors have proven efficacious in inducing immune responses after direct injection in vivo. Among them, recombinant, self-inactivating lentiviral vectors are very attractive delivery systems, as they are able to efficiently transduce into and express foreign genes in a wide variety of mammalian cells. A self-inactivating lentiviral vector was evaluated for the delivery of human immunodeficiency virus 1 (HIV-1) envelope sequences in mice in order to elicit specific immune responses. With this aim, BALB/c mice were immunized with a single injection of self-inactivating lentiviral vectors carrying either the full-length HIV-1(HXB2) Rev/Env (TY2-IIIBEnv) or the codon-optimized HIV-1(JR-FL) gp120 (TY2-JREnv) coding sequence. Both vectors were able to elicit specific cellular responses efficiently, as measured by gamma interferon ELISPOT and chromium-release assays, upon in vitro stimulation of splenocytes from BALB/c immunized mice. However, only the TY2-JREnv-immunized mice were able to elicit specific humoral responses, measured as anti-gp120 antibody production. These data provide the first evidence that a single, direct, in vivo administration of a lentiviral vector encoding a viral gene might represent a useful strategy for vaccine development.

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production.

BACKGROUND: We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated. METHODS: A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production. RESULTS: We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant. CONCLUSIONS: By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.

Defective rev response element (RRE) and rev gene in HAART treated AIDS patients with discordance between viral load and CD4+ T-cell counts.

BACKGROUND: Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDS patients. However, after variable period of treatment, many AIDS patients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious. OBJECTIVE: The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDS patients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts. STUDY DESIGN: The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime. RESULTS AND CONCLUSION: Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.

System-level identification of transcriptional circuits underlying mammalian circadian clocks.

Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

Spliced human immunodeficiency virus type 1 RNA is reverse transcribed into cDNA within infected cells.

Both the full-length and spliced RNA species of HIV-1 possess the necessary cis-acting elements including the primer binding site (PBS), the polypurine tract (PPT), as well as the 5' R and 3' R regions that are needed for their conversion to double-stranded cDNA through reverse transcription. Since measurable amounts of spliced viral RNA molecules can be detected within virus particles, we have examined the potential for reverse transcription of such virion-associated spliced viral RNA upon infection of permissive cells. Analysis of viral cDNA species by PCR and DNA sequencing not only led to the identification of viral DNA molecules that were reverse transcribed from full-length viral RNA, but also DNA molecules that displayed the same nucleotide sequences as those found in spliced viral RNA, except that the former harbored the complete long terminal repeats (LTR), a feature that distinguishes proviral DNA from viral genomic RNA. Further studies revealed various types of cDNA species that resemble the spliced viral RNA encoding each of the env, tat, rev, or nef genes, of which the nef cDNA represents the majority. Therefore, spliced HIV-1 RNA molecules must have been reverse transcribed along with full-length viral RNA during infection.

Rational development of a HIV-1 gene therapy vector.

BACKGROUND: HIV-1 provides an attractive option as the basis for gene transfer vectors due to its ability to stably transduce non-cycling cell populations. In order to fully utilise the promise of HIV-1 as a vector it is important that the effects of viral cis sequence elements on vector function are carefully delineated. METHODS: In this study we have systematically evaluated the effect of various cis elements from the HIV-1 YU-2 genome that have been implicated as either affecting vector performance, or HIV-1 replication, on the efficiency of vector production (titre and infectivity). As a measure of the relative safety of vectors their propensity to inadvertently transfer the gagpol gene to transduced cells was assessed. RESULTS: Sequences that were found to increase vector titre were from the 5' end of the gag gene, from the 5' and 3' ends of the env gene, from immediately upstream of the polypurine tract, and the central polypurine tract. The substitution of the HIV-1 RRE with heterologous RNA transport elements, or the deletion of the RRE, resulted in greatly reduced vector titres. RNA analysis suggested that the role of the Rev/RRE system extends beyond simply acting as an RNA nuclear export signal. The relative safety of different vector designs was compared and an optimal construct selected. CONCLUSIONS: Based on our results we have constructed a vector that is both more efficient, and has better safety characteristics, than the widely used pHR' HIV-1 vector construct.

Contribution of the rev gene to the immunogenicity of DNA vaccines targeting the envelope glycoprotein of HIV.

BACKGROUND: The Rev protein of HIV plays a critical role in the export of viral mRNA from the nucleus to the cytoplasm of infected cells. This work examines the effect of introducing rev into a DNA vaccine encoding the Env protein of HIV, and compares the activity of env genes regulated by CMV versus CAG promoters. METHODS: The HIV Env gp160 encoding gene with or without the rev gene was subcloned into a CMV promoter or a CAG promoter-driven expression plasmid. The Env protein expression of the plasmids was examined in vitro and the HIV-specific immunity was explored in BALB/c mice by an intramuscular route. The immune mice were intraperitoneally challenged with an HIV Env-expression vaccinia virus. RESULTS: Results indicate that the CAG promoter induces significantly higher levels of Env expression, and better immune responses, than the CMV promoter. Incorporating the rev gene into these plasmids further boosts antigen expression and immunogenicity. Indeed, vaccination with the pCAGrev/env or pCMVrev/env plasmid resulted in 1000-fold lower viral load than that with pCMVenv when the mice were challenged with an Env-expressing vaccinia virus. CONCLUSIONS: Incorporating rev into a DNA vaccine significantly increases the level of expression and immunogenicity of a co-expressed env gene, and that protective efficacy is further improved by utilizing a pCAG promoter.

Inhibiting AIDS in the central nervous system: gene delivery to protect neurons from HIV.

Gene therapy to treat primary and secondary CNS diseases, including neuro-AIDS, has not yet been effective. New approaches to delivering therapeutic genes to the central nervous system are therefore required. Recombinant SV40 vectors (rSV40) transduce both dividing and quiescent cells efficiently, and so we tested them for their ability to deliver anti-HIV-1 transgenes to terminally differentiated human NT2-derived neurons (NT2-N). These vectors transduced >95% of immature as well as mature human neurons efficiently, without detectable toxicity and without requiring selection. rSV40 gene delivery was stable to retinoic acid-induced neuronal differentiation. The rSV40 vectors used in these studies, SV(RevM10) and SV(AT), respectively carried the cDNAs for RevM10, a trans-dominant mutant of HIV-1 Rev, and human alpha1-antitrypsin. As measured by HIV-1 p24 antigen assays and by immunostaining for gp120, NT2-N treated with these vectors strongly resisted challenge with different strains of HIV-1. Protection from HIV replication and HIV-induced cytotoxicity was conferred by SV(AT) and SV(RevM10) and remained constant throughout retinoic acid-induced neuronal differentiation and for the duration of these studies (> or =11 weeks). rSV40 transduction of human neurons might therefore be a practicable approach to gene delivery for the treatment of CNS diseases, including neuro-AIDS.

Development of an Ad7 cosmid system and generation of an Ad7deltaE1deltaE3HIV(MN) env/rev recombinant virus.

A strategy to circumvent immune responses to adenovirus (Ad) resulting from natural infection or repeated vector administrations involves sequential use of vectors from different Ad serotypes. To further develop an Ad-HIV recombinant AIDS vaccine approach, a replication-defective recombinant Ad from a non-subgroup C virus was required. Using a cosmid system, we generated an Ad7deltaE1deltaE3HIV(MN) env/rev recombinant virus and compared expression of the inserted HIV genes with a similarly constructed replication-competent Ad7deltaE3HIV(MN)env/rev recombinant. Ad7deltaE1deltaE3HIV(MN)env/rev expressed both HIV env and rev gene products. The envelope protein was correctly processed and functional, mediating syncytia formation of Ad7deltaE1deltaE3HIV(MN) env/rev-infected cells and CD4(+) T lymphocytes. Ad7deltaE1deltaE3HIV(MN)env/rev could be amplified on 293-ORF6 cells, containing the E4 ORF6 gene, shown earlier to support production of an Ad7 vector lacking the E1a gene. The utility of this cell line is now extended to the production of replication-defective Ad7 recombinants lacking E1a, E1b, and protein IX genes. Sequential immunizations with Ad-HIV recombinants based in different Ad serotypes have been shown to effectively elicit both humoral and cellular HIV-specific immune responses. The recombinant Ad7deltaE1deltaE3HIV(MN)env/rev will be useful in such AIDS vaccine strategies. Further, these studies have created new cosmid vectors that can be applied to generation of single- or double-deleted Ad7 recombinants with foreign genes inserted into the E1 and/or E3 regions.

Characterization of the caprine arthritis encephalitis virus (CAEV) rev N-terminal elements required for efficient interaction with the RRE.

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C.