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.     

Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.

HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.     

Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids

Interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region within env mRNA (termed RRE) mediates the export of virus structural mRNAs from the nucleus to the cytoplasm. We show that the region encompassing the basic stretch of amino acids is essential for the ability of Rev to bind to RRE RNA and function in vivo. By use of a functional truncated Rev protein in conjunction with authentic Rev, effects on gel mobilities of the Rev-RRE RNA complex attributable to multimerization of Rev protein were observed. Rev proteins, unable to multimerize, failed to bind RRE RNA. Identification of Rev mutants capable of forming multimers, but unable to bind RRE RNA, suggests that the multimerization and RNA-binding domains can be distinguished and that multimerization is likely a prerequisite for formation of the RRE RNA-binding site. A mutant Rev protein, shown previously to function as a trans-dominant inhibitor of Rev function, bound to RRE RNA as a multimer to a similar extent as wild-type Rev. This observation is consistent with the hypothesis that regulation of HIV gene expression by Rev involves the interaction with cellular factors and that the trans-dominant Rev is probably defective in this function.     

A nucleolar localizing Rev binding element inhibits HIV replication

The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment.     

Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs.

The effect of Rev on cytoplasmic accumulation of the singly spliced human immunodeficiency virus type 1 (HIV-1) vif, vpr, and env/vpu RNAs was examined by using a quantitative RNA polymerase chain reaction (PCR) analysis following transfection of complete proviral molecular clones into lymphoid cells. Previously published studies using subgenomic env constructs in nonlymphoid cell types concluded that Rev was necessary for cytoplasmic accumulation of high levels of unspliced env RNA and that, by analogy, Rev must be necessary for the cytoplasmic accumulation of all HIV-1 RNAs that contain the Rev-responsive element (RRE). We confirm those results in COS cells. Unexpectedly, in lymphoid cells, we find that although Rev acts somewhat to increase the cytoplasmic level of full-length HIV-1 RNA, Rev has little or no effect on cytoplasmic accumulation of singly spliced HIV-1 RNAs. However, Env protein expression was greatly reduced in the absence of Rev. Analysis of the cytoplasmic RNA revealed that in the absence of Rev or the RRE, the cytoplasmic vif, vpr, and env/vpu 2 RNAs were not associated with polysomes but with a complex of 40S-80S in size. Consequently, efficient expression of the Vif, Vpr, Vpu, and Env proteins from these RNAs is dependent on Rev. These results exclude a mechanism whereby the sole function of Rev is simply to export RNAs from nucleus to cytoplasm. We discuss other models to take into account the dependence on Rev for efficient translation of cytoplasmic HIV-1 RNAs.     

Identification of nucleolus localization signal of betanodavirus GGNNV protein alpha

Betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis viruses (GGNNV) protein alpha, a virus capsid protein, was detected in both nucleolus and cytoplasm of infected cells of Asian sea bass (SB) and transfected cells of SB and Cos-7 with pcDNA3.1/RNA2. To study its subcellular localization, ORF of protein alpha with 338 aa was fused with enhanced green fluorescent protein (EGFP) gene and was detected in transfected cells in the absence of other viral proteins. In both SB and Cos-7 cells, protein alpha was found to localize EGFP to the nucleolus and cytoplasm. Deletion mutants of protein alpha indicated that N-terminal 43 amino acid residues were required to import EGFP-alpha protein into the nucleolus. Further deletions within the 43 amino acid backbone, EGFP/33aa(1-33) and EGFP/30aa(14-43), localized to the nucleolus, suggesting that the 20 amino acids from 14 to 33 of protein alpha were the domain of nucleolus localization. To further determine the nucleolus targeting sequence, deletion mutations within the 20 amino acids of protein alpha were constructed. It was found that the deletion of (23)RRR(25), (29)RRR(31), or (23)RRRANNRRR(31) prevented the accumulation of EGFP fusion proteins into the nucleolus, demonstrating that (23)RRRANNRRR(31) contain the signal required for nucleolar localization. A similar distribution pattern of localization of protein alpha and its deletion mutants in SB and Cos-7 cells suggested that N-terminal residues of protein alpha (23)RRRANNRRR(31) constitute a nucleolus localization signal that functions in both fish and mammalian cells.     

Subcellular localization of rev-gene product in visna virus-infected cells

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.     

A pre-translational defect in a case of human mu heavy chain disease

A patient (BW) was studied with Mu heavy chain disease (mu HCD) in whom a leukemic B-cell clone secreted a shortened monoclonal mu chain without associated light chain. The cells did, however, produce a normal-sized kappa light chain that was detected as urinary Bence-Jones protein. The cytoplasmic and secreted monomeric mu chain had an approximate mol. wt of 58,000. Radiochemical sequence analysis of the biosynthetically labelled mu chain revealed a protein that lacked the entire variable region. The sequence initiated at amino acid position 5 within the first constant region domain (CH1) of C mu. The primary in vitro translation product, the cytoplasmic and secreted proteins were all similarly truncated, thereby excluding extensive postsynthetic degradation. The mu RNA, that directed the synthesis of the truncated mu protein, was about 350 bp smaller than the normal mu RNA. Furthermore, by primer extension analysis it was possible to localize this deletion in the mu RNA to a region 5' of CH1. Thus, a defect at the level of Ig gene structure/assembly that deletes coding information or results in aberrant RNA processing must be responsible for the truncated mu HCD protein BW.     

Mutants in a conserved region near the carboxy-terminus of HIV-1 Rev identify functionally important residues and exhibit a dominant negative phenotype

The rev protein (Rev) of human immunodeficiency virus increases the cytoplasmic expression of viral structural gene mRNAs. We had previously reported the existence of a region (residues 73-98) near the carboxy-terminus in HIV-1 Rev essential for its function. To further define the structural elements in this region, we examined the effects of substitution mutations in highly conserved residues in this region, between amino acids 75-81, on Rev function. Mutations in Pro76-77 and Arg80 retained Rev function, whereas those in Leu75 and Leu81 abolished Rev activity and exhibited trans-dominant suppression of wt Rev function. The Leu81 mutation, in particular, exhibited an efficient dominant negative phenotype. Leu75 and Leu81 thus appear to define residues essential to the Rev "effector" function.     

An upstream regulatory sequence stimulates expression of the perfringolysin O gene of Clostridium perfringens.

The structural gene for perfringolysin O (pfoA), a thiol-activated hemolysin of Clostridium perfringens, was cloned into Escherichia coli JM109 on a 4.6-kilobase (kb) EcoRI-NdeI fragment which contained the 1.7-kb pfoA gene and an upstream 2.9-kb region. An E. coli strain transformed by this plasmid produced 20-fold more perfringolysin O than a strain containing only the 1.7-kb pfoA gene. The stimulatory effect of the upstream region on in vivo expression of the pfoA gene was further analyzed by using a set of deletion mutants. Stimulation was still observed with a 3.9-kb fragment, but stimulation was not observed with fragments that were 3.6 kb or less long, indicating that the upstream region between 3.9 and 1.7 kb was involved in activation of pfoA gene expression. Nucleotide sequencing showed that this region contained one open reading frame (pfoR) coding for 343 amino acids. The deduced amino acid sequence of pfoR possesses several motifs that are characteristic of DNA-binding proteins. When a region coding for a helix-turn-helix, one of the most important motifs of DNA-binding proteins, was deleted within pfoR, stimulation was completely abolished. These results indicate that pfoR positively controls expression of the pfoA gene.     

Molecular cloning characterization and expression of porcine immunoreceptor SIRPalpha

SWC3 is a porcine CD that has been the reference marker of myeloid lineage. It is expressed in every myelomonocytic cell from early bone marrow precursors. We have identified the molecule recognized by anti-SWC3 antibodies as a member of the signal-regulatory proteins (SIRPs)alpha family. Here, we describe the cloning of a cDNA coding for a porcine SIRPalpha protein. The sequence is 2470 nucleotides long and contains an open reading frame encoding a 507 amino acid sequence. The predicted polypeptide was composed of a 30 amino acids putative signal peptide, a 342 amino acid extracellular region, a 23 amino acid transmembrane segment and a 112 amino acid cytoplasmic domain. Analysis of the sequence reveals a high degree of homology with known SIRPs in other species, being easily identified the three extracellular Ig type domains and two cytoplasmic ITIM motifs characteristic of this molecule. The gene coding for porcine SIRPalpha has been mapped to porcine chromosome 17, in a region syntenic to the human chromosome 20 where SIRP genes have been mapped. During the analysis of SIRP gene expression in tissues by RT-PCR, we noticed the existence of a shorter mRNA, and cloned the corresponding cDNA. This coded for a splicing variant of SIRPalpha that lacked the two membrane proximal Ig domains. In transfection experiments, we have been able to show that anti-SWC3 antibodies recognize both forms of the molecule, mapping the SWC3 epitopes to the N-terminal IgV type domain.     

Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization.

The Escherichia coli mannitol permease (EIIMtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out the concomitant transport and phosphorylation of D-mannitol. Previous studies indicated that there are ca. 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm. In the present study, an analysis of C-terminally truncated EIIMtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at > or = 50% the amount of the intact protein. However, deletion proteins smaller than ca. 34 kDa were present in the membrane at only about 20% the amount of the intact protein. We also constructed a plasmid that encodes the first 43 amino acid residues of ELLMtl fused to residues 378 to 637 (the C-terminal domain). The corresponding protein was associated with the cytoplasmic membrane. These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain. Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed. Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein. These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization.     

Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein

The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation.     

Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 that is capable of producing virus-like particles

Ectopic expression of the structural protein Pr55^Gag of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5′-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3′-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55^Gag in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55^Gag derived from this JEV replicon RNA appeared to be increased by 3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55^Gag proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55^Gag could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55^Gag by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.     

Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef,Tat and Rev in acutely and chronically infected lymphoid cell lines

Summary Information concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6–9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12–48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.