The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.     

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.     

Avian reticuloendotheliosis virus strain A and spleen necrosis virus do not infect human cells

Spleen necrosis virus (SNV) and Reticuloendotheliosis virus strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769-4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301-310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.     

Anti-Rex aptamers as mimics of the Rex-binding element

RNA molecules that bind tightly and specifically to a Rex fusion protein have been isolated from a conformationally constrained pool of random sequence RNAs. The anti-Rex aptamers effectively mimic several features of the wild-type Rex-binding element (XBE). The highest-affinity aptamers effectively compete with the wild-type XBE for binding to the RNA-binding domain of Rex, an arginine-rich motif (ARM), but do not bind to the functionally analogous Rev protein or its ARM. However, characteristic sequence and structural motifs found in some of the anti-Rex aptamers may provide insights into how the Rex protein can interact with other viral RNAs, such as the Rev-responsive element. The anti-Rex aptamers can functionally substitute for the XBE in vivo, a result which supports a previously proposed model for mRNA transport in which the viral genome serves as a platform for assembling a nucleoprotein complex that can co-opt the cellular transport apparatus. Overall, these studies suggest that anti-Rex aptamers may serve as RNA decoys of the Rex protein.     

HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA

We have used an iterative in vitro genetic selection to identify the important structural features of the viral RNA element bound by the Rev protein of human immunodeficiency virus type 1 (HIV-1). Functional Rev-binding RNAs were selected from a pool of 10(13) variants of the wild-type Rev-binding domain. Bases conserved among the binding species define a 20 nucleotide core binding element. Covariation of some of these conserved bases indicates that the Rev-binding element is a stem-bulge-stem with a G:G base pair in the bulge. Mutational studies show that this non-Watson-Crick base pair is required for Rev binding in vitro and Rev responsiveness in vivo. We propose that the G:G base pair distorts the sugar-phosphate backbone of viral RNA and that this distortion is a critical determinant of recognition by Rev.     

The receptor for the subgroup A avian leukosis-sarcoma viruses binds to subgroup A but not to subgroup C envelope glycoprotein.

The putative subgroup A avian leukosis-sarcoma virus (ALSV) receptor (Tva) was recently cloned by gene transfer (P. Bates, J. A. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Susceptibility to infection by subgroup A ALSV is conferred on cells upon transfection with cDNAs encoding tva. The hypothesis that tva encodes a specific receptor for subgroup A ALSV predicts that the Tva protein should bind to subgroup A, but not to subgroup C, envelope glycoprotein. In this study, we examined this prediction by using several biochemical assays. We established stable NIH 3T3 cell lines expressing either Tva, the subgroup A envelope glycoprotein (Env-A), or the subgroup C envelop glycoprotein (Env-C) and used them in conjunction with soluble forms of these molecules to demonstrate specific binding. When cell lysates containing Tva were mixed with lysates of either Env-A or Env-C, an immunoprecipitable complex formed between Tva and Env-A but not between Tva and Env-C. A soluble, oligomeric form, of Env-A, not Env-C, binds to cells expressing Tva. Reciprocally, a secreted form of Tva can bind to cells expressing Env-A but not to cells expressing Env-C. A specific and stable complex formed between soluble Env-A and secreted Tva as demonstrated by sucrose density gradient centrifugation. Thus, by three kinds of assays, Tva appears to bind specifically to Env-A, which is consistent with genetic evidence that it serves as the cell surface receptor of subgroup A ALSV and the main determinant of subgroup specificity.     

Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA.

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.     

Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules.

The entry of human immunodeficiency virus type 1 into cells proceeds via a fusion mechanism that is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. Species- and tissue-specific restrictions to viral entry suggested the participation of additional membrane components in the postbinding fusion events. In a previous study (H. Golding, J. Manischewitz, L. Vujcic, R. Blumenthal, and D. Dimitrov, J. Virol. 68:1962-1968, 1994), it was found that phorbol myristate acetate (PMA) inhibits human immunodeficiency virus type 1 envelope-mediated cell fusion by inducing down modulation of an accessory component(s) in the CD4-expressing cells. The fusion inhibition was seen in a variety of cells, including T-cell transfectants expressing engineered CD4 receptors (CD4.401 and CD4.CD8) which are not susceptible to down modulation by PMA treatment. In the current study, it was found that preincubation of A2.01.CD4.401 cells with soluble monomeric gp120 for 1 h at 37 degrees C primed them for PMA-induced down modulation (up to 70%) of the tailless CD4 receptors. The gp120-priming effect was temperature dependent, and the down modulation may have occurred via clathrin-coated pits. Importantly, nonhuman cell lines expressing tailless CD4 molecules did not down modulate their CD4 receptors under the same conditions. The gp120-dependent PMA-induced down modulation of tailless CD4 receptors could be efficiently blocked by the human monoclonal antibodies 48D and 17B, which bind with increased avidity to gp120 that was previously bound to CD4 (M. Thali, J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski, J. Virol. 67:3978-3988, 1993). These findings suggest that gp120 binding to cellular CD4 receptors induces conformational changes leading to association of the gp120-CD4 complexes with accessory transmembrane molecules that are susceptible to PMA-induced down modulation and can target the virions to clathrin-coated pits.     

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.     

RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.     

Complete Genome Sequence of T4-Like Escherichia coli Bacteriophage HX01

Phage T4 is among the best-characterized biological systems (S. Kanamaru and F. Arisaka, Seikagaku 74:131-135, 2002; E. S. Miller et al., Microbiol. Mol. Biol. Rev. 67:86-156, 2003; W. B. Wood and H. R. Revel, Bacteriol. Rev. 40:847-868, 1976). To date, several genomes of T4-like bacteriophages are available in public databases but without any APEC bacteriophages (H. Jiang et al., Arch. Virol. 156:1489-1492, 2011; L. Kaliniene, V. Klausa, A. Zajanckauskaite, R. Nivinskas, and L. Truncaite, Arch. Virol. 156:1913-1916, 2011; J. H. Kim et al., Vet. Microbiol. 157:164-171, 2012; W. C. Liao et al., J. Virol. 85:6567-6578, 2011). We isolated a bacteriophage from a duck factory, named HX01, that infects avian pathogenic Escherichia coli (APEC). Sequence and morphological analyses revealed that phage HX01 is a T4-like bacteriophage and belongs to the family Myoviridae. Here, we announce the complete genome sequence of phage HX01 and report the results of our analysis.     

Analysis of the subgroup A avian sarcoma and leukosis virus receptor: the 40-residue, cysteine-rich, low-density lipoprotein receptor repeat motif of Tva is sufficient to mediate viral entry.

The genes encoding the receptor for subgroup A Rous sarcoma viruses (tva) were recently cloned from both chicken and quail cells (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Previous work suggested that only the extracellular domain of Tva interacts with the virus (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). Tva is a small membrane-associated protein containing in its extracellular domain a 40-amino-acid region which is closely related to the low-density lipoprotein receptor (LDLR) repeat motif. To determine the region of the Tva extracellular domain responsible for viral receptor function, we created chimeric proteins containing various regions of the Tva extracellular domain fused with a murine CD8 membrane anchor. Analysis of these proteins demonstrates that any chimera containing the Tva LDLR repeat motif can specifically bind the envelope protein of subgroup A avian sarcoma and leukosis viruses. Furthermore, NIH 3T3 cell lines expressing these chimeric proteins were efficiently infected by subgroup A avian sarcoma and leukosis virus vectors. Our results demonstrate that the 40-residue-long LDLR repeat motif of Tva is responsible for viral receptor function.     

Interaction between the dominant negative mutant and the wild-type envelope proteins of Friend murine leukemia virus.

Interaction between the previously obtained dominant negative mutant, referred to as fcr (T. Matano, T. Odawara, M. Ohshima, H. Yoshikura, and A. Iwamoto, J. Virol. 67:2026-2033, 1993), and the wild-type envelope proteins (Env) of Friend murine leukemia virus was examined. The wild-type Env was bound to the fcr mutant Env and trapped in the endoplasmic reticulum. The virus receptor was not involved in this interaction.     

Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line.

We constructed recombinant reticuloendotheliosis virus (Rev)-derived and murine leukemia virus-derived vectors to characterize the specificity of packaging retroviral RNAs in Rev proteins. Using this approach, we further localized the Rev encapsidation sequence (E) to a 144-nucleotide region and determined that there are sequences in both the 5' and 3' halves of this region which are necessary in cis for viral replication. We found that the Rev E, like the murine leukemia virus E (psi), is position independent (R. Mann and D. Baltimore, J. Virol. 54:401-407, 1986). Also, a 156-nucleotide region of the Rev intron enhanced replication in a cis-acting fashion in the presence, but not in the absence, of helper virus. Finally, we showed that packaging of E- and heterologous retroviral genomes occurred efficiently in the Rev helper cell in the absence of competing E-containing (E+) viral RNAs.     

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.     

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.     

A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA

Many ribonucleoprotein (RNP) complexes assemble into large, organized structures in which protein subunits are positioned by interactions with RNA and other proteins. Here we demonstrate that HIV Rev, constrained in size by a limited viral genome, also forms an organized RNP by assembling a homo-oligomer on the Rev response element (RRE) RNA. Rev subunits bind cooperatively to discrete RNA sites using an oligomerization domain and an adaptable protein-RNA interface, forming a complex with 500-fold higher affinity than the tightest single interaction. High-affinity binding correlates strongly with RNA export activity. Rev utilizes different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites, including the well-characterized stem IIB site and an additional site in stem IA. We propose that adaptable RNA-binding surfaces allow the Rev oligomer to assemble economically into a discrete, stable RNP and provide a mechanistic role for Rev oligomerization during the HIV life cycle.