Antimicrobial properties of nuclear diffusion inhibitory signal of HIV-1 Rev.

Nuclear Diffusion Inhibitory Signal (NIS) has been identified in human immunodeficiency virus type 1 (HIV-1) Rev as a nuclear signal peptide which modulates nucleocytoplasmic protein trafficking and intracellular stability of the HIV-1 Rev. In this study, it was discovered that antimicrobial properties are inherent in the NIS. This is a significant finding that the NIS, which does not exist solely for self defense, in fact possesses antimicrobial properties.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.

The HIV-1 transactivator protein Tat is a potent inducer of the human DNA repair enzyme beta-polymerase.

OBJECTIVE: This study examines the effects of the HIV-1 regulatory proteins, Tat and Rev, on the expression of the DNA polymerase beta (beta-pol) gene, which encodes a key protein in the DNA base-excision repair pathway. The rationale for these experiments is to examine the potential involvement of base-excision repair protein deregulation in HIV-1-related lymphomas. DESIGN: Expression of beta-pol mRNA was examined in AIDS-related lymphomas and non-AIDS-related lymphomas and as a function of HIV-1 infection of B cells in culture. The effect of Tat or Rev over-expression on beta-pol promoter expression was tested by transient co-transfection assays with a beta-pol promoter reporter plasmid and a Tat or Rev over-expression plasmid. METHODS: Northern blot analysis was used to quantitate beta-pol expression in lymphoma and cells. Raji cells were co-transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid and a plasmid over-expressing Tat or Rev. CAT activity was measured in transfected cells. RESULTS: beta-Pol mRNA was > 10-fold higher in AIDS-related than in non-AIDS B-lineage lymphomas. beta-Pol expression was up-regulated in a B-cell line upon infection with HIV-1, and increased in Raji cells upon recombinant expression of the Tat gene. The beta-pol promoter was transactivated (fourfold induction) by Tat, but not by Rev. Tat-dependent transactivation required a binding site for the transcription factor Sp1 in the beta-pol promoter. CONCLUSION: These results suggest that HIV-1 Tat can interact with cellular transcription factors to increase the steady-state level of beta-pol in B cells. Tat-mediated induction of beta-pol may alter DNA stability in AIDS-related lymphomas.

Inhibition of HIV-1 replication and infectivity by expression of a fusion protein, VPR-anti-integrase single-chain variable fragment (SFv): intravirion molecular therapies.

OBJECTIVES: To deliver antiretroviral agents or other foreign proteins into progeny virions and evaluate their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication. STUDY DESIGN/METHODS: HIV-1 encodes proteins in addition to gag, pol, and env, some of which are packaged into virus particles. One essential retroviral enzyme is integrase (IN), which has been used as a target for developing agents that inhibit virus replication. In previous studies, we demonstrated that intracellular expression of single-chain variable antibody fragments (SFvs), which bind to IN, results in resistance to productive HIV-1 infection in T-lymphocytic cells. Because the highly conserved accessory HIV-1 Vpr protein can be packaged within virions in quantities similar to those of the major structural proteins, this primate lentiviral protein may be used as a fusion partner to deliver antiviral agents or other foreign proteins into progeny virions. In these studies, the fusion proteins Vpr-chloramphenicol acetyl transferase (CAT) and Vpr-SFv-IN have been developed. Stable transfectants expressing these fusion proteins were generated from PA317 cells and SupT1 T-lymphocytic cells and analyzed using immunofluorescence microscopy. After challenge of SupT1 cells with HIV-1, p24 antigen expression was evaluated. The incorporation of these fusion proteins were evaluated by immunoprecipitation of virions using a Vpr antibody. RESULTS: Expression of the fusion proteins was confirmed by immunofluorescent staining in PA317 cells transfected with the plasmids expressing Vpr-CAT and Vpr-SFv-IN proteins. Stable transfectants expressing these fusion proteins were generated from SupT1 T-lymphocytic cells. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression, was inhibited in cells expressing Vpr-SFv-IN. It was demonstrated that Vpr-chloramphenicol acetyl transferase (Vpr-CAT and Vpr-SFv-IN proteins can be efficiently packaged into the virions and that Vpr-SFv-IN also decreases the infectivity of virions into which it is encapsidated. CONCLUSIONS: An anti-integrase single-chain variable fragment moiety can be delivered into HIV-1 virions by fusing it to Vpr. Vpr-SFv-IN decreases HIV-1 production in human T-lymphocytic cells. The benefits of "intravirion" gene therapy include immunization of target cells as well as decreasing infectivity of HIV-1 virions harboring the fusion construct. Thus, this approach to anti-HIV-1 molecular therapies has the potential to increase inhibitory effects against HIV-1 replication and virion spread.

The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element.

Small nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus.

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression.

The HIV-1 encoded regulatory Rev protein acts to selectively increase the cytoplasmic concentration of incompletely spliced viral mRNAs through interaction with the Rev responsive element (RRE). In addition, the Rev activation domain, believed to be a nuclear export sequence, has been shown to modulate the export of non-RRE containing RNAs (e.g. 5S rRNA, splicesomal U snRNAs). Recent evidence suggests Rev activity depends on interactions with cellular cofactors, leading to speculation that Rev utilizes a cellular RNA and/or a protein export pathway. Rev interactions with cellular cofactors could lead to sequestration of those cofactors from normal cellular activities, suggesting potential Rev effects on cellular gene products and their resultant activity. We have examined the role of Rev in modulating the expression of cellular gene products. Through transient cotransfection assays, we observed a consistent and significant decrease in the levels of luciferase and B-galactosidase activity in the presence of a Rev expressing construct. Cell fractionation studies demonstrated the nuclear retention of the luciferase gene transcripts. Surprisingly, similar effects were observed on constitutively expressed RNAs such as gamma-actin transcripts, and the 18S and 28S rRNAs. These results suggest Rev can disrupt the nuclear export of multiple classes of RNAs.

Interleukin-10 gene expression induced by HIV-1 Tat and Rev in the cells of HIV-1 infected individuals.

The role of cytokines in the regulation and function of the immune system is of great importance. In human immunodeficiency virus (HIV) infection, with progressive deterioration of cell-mediated immune response, cytokines are dysregulated. We have therefore investigated cytokine mRNA expression in type-1 and type-2 helper T cells of HIV-seropositive (HIV+) individuals, stimulated with mitogen (leukoagglutinin) and HIV-1 Tat and Rev peptides, previously found to induce proliferative T-cell responses in these individuals. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect interleukin 2 (IL-2), interferon gamma (IFN-gamma), IL-4, and IL-10 mRNAs. There was no difference in the mRNA expression of these cytokines when the cells of HIV-infected or noninfected individuals were polyclonally stimulated with the mitogen, as all cytokine mRNAs were detected in both groups. Baseline cytokine expression of unstimulated cells was, however, different in these two groups: the cells of HIV+ persons did not show comparable expression of mRNAs to HIV-seronegative (HIV+) individuals. When the cells of HIV+ individuals were stimulated with the peptides, 70% of the cases showed IL-10 mRNA expression, 20% IFN-gamma, and 10% IL-2, with no detection of IL-4 mRNA in any of the cases. Our results thus show that HIV-specific T-cell antigens induce production of IL-10 in HIV-infected individuals. The increase in IL-10 demonstrated here may have a role in hyperactivation of B cells, as well as in immunosuppression of T cells often seen in HIV-infected individuals.

Elements distinct from human immunodeficiency virus type 1 splice sites are responsible for the Rev dependence of env mRNA.

In the absence of the viral regulatory protein Rev, the human immunodeficiency virus type 1 gag/pol and env mRNAs are inefficiently expressed, since nucleocytoplasmic transport, stability, and polysomal loading are impaired. It has been suggested that splicing is necessary for Rev function and that the low expression of the unspliced and intermediate spliced mRNAs in the absence of Rev is associated with specific splice sites. Previous studies identified distinct RNA elements within the gag/pol region responsible for low expression that are not associated with splice sites. Here we study the determinants for Rev dependence of the authentic env mRNA. We demonstrate that upon removal of all the utilized splice sites, the env mRNA is still Rev dependent and Rev responsive for expression in human cells. We have identified several regions within the env mRNA that inhibit expression of a gag-env hybrid mRNA. Elimination of one of these elements, located within the Rev-responsive element, did not result in virus expression, supporting our model that several independently acting elements are responsible for the downregulatory effect. By analogy to the RNA elements within the gag/pol region, we propose that elements unrelated to utilized splice sites are responsible for the posttranscriptional regulation of env mRNA.

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.

Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron.

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regulation of HIV env expression is critically dependent on whether the env region is defined as an intron. This strengthens the hypothesis that Rev interacts with components of the splicing machinery to release splicing factors and enable export of the mRNA before splicing occurs.

Cytotoxic activity of rev protein of human immunodeficiency virus type 1 by nucleolar dysfunction.

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells. However, similarly expressed rex protein (Rex) of the human T-cell leukemia virus type I, which is a functional homologue to Rev, did not affect nucleolar structure and function. Rev expression resulted in cell death with nucleolar destruction in an inducible cell line. Analysis of Rev mutants revealed that both the nucleolar targeting signal of Rev and the multimerization domain are prerequisites to the nucleolar disintegration by Rev. Human T-cells acutely infected with HIV-1 contained nucleoli which were deformed and filled with Rev, but chronically infected cells had intact nucleoli. Involvement of Rev in cytopathic effects in HIV-1 infection is discussed.

The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly.

Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev binds to a cis-acting Rev-responsive element (RRE) located within the env region of HIV-1. It has previously been shown that a 17-amino-acid peptide, corresponding to the basic domain of Rev, specifically inhibited in vitro the splicing of mRNAs containing the RRE. In this reaction, the peptide acts after an ATP-dependent step in the spliceosome assembly resulting in an accumulation of a 45-50S splicing-deficient complex. Characterization of this complex revealed that the basic domain of Rev does not interfere with U1 small nuclear ribonucleoprotein binding but blocks the entry of U4, U5, and U6 small nuclear RNAs into the spliceosome. Binding of U2 small nuclear ribonucleoprotein was partially inhibited. The critical nature of the oligomeric structure of RRE has been investigated both in vitro and in vivo. Reporter genes that contained one, three, or six repeated-monomer high-affinity Rev binding sites (IIB) within an intron yielded a correlation among the oligomeric state of bound Rev; inhibition of splicing; ability to block the assembly of U4, U5, and U6 small nuclear RNAs in the spliceosome in vitro; and level of Rev response in vivo.