Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake.

A Gram-negative, non-motile, neutrophilic, rod-shaped, extremely halophilic archaeon, designated strain BG-1(T), was isolated from a salt lake, Lake Bagaejinnor, in Inner Mongolia, China. Strain BG-1(T) was able to grow at 25-55 degrees C, required at least 2.5 M NaCl for growth (with an optimum at 3.4 M NaCl) and grew at pH 6.0-9.0 (with an optimum at pH 7.5). Hypotonic treatment with less than 2.0 M NaCl caused cell lysis. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence positioned the isolate within the genus Halorubrum in the family Halobacteriaceae. Strain BG-1(T) was most closely related to Halorubrum aidingense 31-hong(T) (98.8% sequence similarity), Halorubrum saccharovorum NCIMB 2081(T) (98.6%), Halorubrum lacusprofundi ACAM 34(T) (98.6%) and Halorubrum lipolyticum 9-3(T) (98.4%). However, values for DNA-DNA hybridization between strain BG-1(T) and the most closely related members of the genus Halorubrum were below 40%. Analysis of the polar lipids of strain BG-1(T) revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid found in neutrophilic species of the genus Halorubrum. The G+C content of the genomic DNA was 69.4 mol% (T(m)). Comparison of the phenotypic characteristics of the strain with those of Halorubrum species supported the conclusion that BG-1(T) represents a novel species within this genus, for which the name Halorubrum kocurii sp. nov. is proposed. The type strain is BG-1(T) (=CECT 7322(T) =CGMCC 1.7018(T) =JCM 14978(T)).

The vaccinia virus E3L protein interacts with SUMO-1 and ribosomal protein L23a in a yeast two hybrid assay.

We report the results of a two-hybrid study which identified clones from a HeLa cDNA library that interact with the vaccinia virus protein E3L. These clones encode the nuclear protein SUMO-1 (also known as PIC-1, sentrin or GMP-1); the cytoplasmic ribosomal protein L23a; and a small peptide sequence of unknown significance.

Nucleotide sequence of the 4.3 kbp BamHI-N fragment of fowlpox virus FP9.

Nucleotide sequence analysis of the 4.3 kbp BamHI-N fragment of the fowlpox virus (FPV) genome revealed that it encodes 7 proteins with homology to vaccinia virus (VV) E11L, E10R, O1L, O3L, I1L, I2L and I3L encoded proteins. No evidence of FPV homolog of VV O2L could be found.

HIV-1 Gag binds specifically to RNA stem-loops in the 5' leader sequence.

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro, with a Kd of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325-362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5' of nucleotide 335 require the presence of additional 3' sequences.

The sequence of a cDNA encoding functional murine C1-inhibitor protein.

The murine C1-inhibitor protein is 482 amino acids long. It consists of an N-terminal domain of 118 amino acids rich in proline and threonine and a serpin domain. The N-terminal domain has 39% identity with the corresponding regions of human and bovine C1 inhibitor.

Antiviruses as therapeutic agents: a mathematical analysis of their potential.

Antiviruses are designed to inhibit virus replication and arrest infections. A particular antivirus derives from a specific virus, on which it depends for propagation. Antiviruses have a natural equivalent in defective interfering particles (DIPs). To obtain design criteria for antiviruses, antivirus therapies for treating virus infections are modelled by a system of equations with continuous dynamics. The results reveal that such therapies can eliminate viruses given either a large but achievable inoculum of antivirus or an outcompeting advantage to the antivirus. Since such therapies are relatively insensitive to many parameters of infections, they may be applicable to many viral diseases.

Production of non-infectious human immunodeficiency virus-like particles which package specifically viral RNA.

A recombinant vector that rapidly produces large amounts of human immunodeficiency virus (HIV) virus-like particles (VLPs) was constructed. This vector lacks LTR sequences and a functional nef gene. The VLPs produced are non-infectious but similar in structure to mature, infectious HIV virions. They package specifically HIV RNAs containing appropriate signals and do not package abundant cellular mRNAs (e.g. actin). In the system described here, efficient particle production and release is decoupled from infection. Use of this VLP system offers many advantages over the study of infectious virions, permitting the expression of mutant phenotypes which interfere with virus infectivity.

Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1.

Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands. A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer. Several models for the formation of the HIV-1 RNA dimer have been proposed. In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop. In the guanine tetrad model interstrand guanine contacts from the dimer. We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms. To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716. To prevent the guanine tetrad dimer forming we changed G819 to U. These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively. All three clones produced infectious virions. Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations. The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation. In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

A study of the dimerization of Rous sarcoma virus RNA in vitro and in vivo.

The Rous sarcoma virus dimer linkage site (DLS) has been located by electron microscopy at position 511 +/- 28 nucleotides. We have studied the dimerization of RNAs encompassing the first 634 nucleotides of Rous sarcoma virus and conclude that there are at least two dimerization signals. One is located between nucleotides 531 and 634 and may involve Watson-Crick pairing of an imperfect inverted repeat. The other signal is located between nucleotides 496 and 530. A tetraguanine sequence at nucleotides 523-526 is required for dimerization of this domain. The guanines are not involved in an identifiable Watson-Crick interaction or in guanine tetrad formation. Either dimerization domain can initiate the dimerization of RNA 1-634. It is possible that these domains are two parts of a single dimerization signal. Interstrand RNA contacts within the virion are not limited to the DLS but occur along the length of the genome. Nascent virions contain monomeric RNA which slowly associates to form an RNA dimer. The limiting step in dimerization is not proteolytic cleavage of the gag precursor because only the mature capsid protein p27 can be detected in these nascent virions.

Designer antiviruses for HIV.

Potentially, antiviruses that interfere with HIV propagation could be used as AIDS therapy. If problems associated with HIV recombination and the dynamics of the interactions between HIV and antivirus can be resolved by an appropriate design, an antivirus might defer or prevent the development of AIDS, and might benefit AIDS patients.

Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA.

Retroviruses package two homologous single-stranded RNA genomes within a gag protein-RNA complex. In mature virion particles, the two RNA strands are thought to associate primarily through direct RNA-RNA interactions, although the structural basis for this stable association is unknown. We now report that a 127-nucleotide (nt) HIV-1NL4-3 RNA fragment (positions 732-858) encompassing the 5' end of the gag gene dimerizes spontaneously under high ionic strength conditions in the absence of any protein cofactor. The HIV-1 RNA dimer is dramatically and specifically stabilized by the monovalent cation potassium. Thermal dissociation of the dimer occurs at 80 degrees C in 100 mM K+ (5 mM Mg2+) but at significantly lower temperatures in the presence of either smaller or larger monovalent cations (100 mM Li+, 40 degrees C; 100 mM Na+, 55 degrees C; 100 mM Cs+, 30 degrees C). Deletion analyses of the 3' end of the 127-nt fragment reveal that an HIV-1 RNA fragment as short as 94 nt (732-825) can dimerize spontaneously, but a further 9-base deletion of the purine-rich sequence, GGGGGAGAA from positions 817 through 825, eliminates dimerization. These experimental results support a model in which HIV-1 RNA dimerizes by forming an interstrand quadruple helix stabilized by guanine (and/or purine)-base tetrads in analogy to the well-known dimerization of telomeric DNA. We speculate that this structure may also mediate the association of genomic HIV-1 RNA in vivo, revealing how RNA itself can achieve the self-recognition required for subsequent genetic recombination.

In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.

Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.

The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the trans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6- to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region.

HIV-1 Rev protein, purified from E. coli, binds specifically to an RNA transcript containing the 223 nucleotide long Rev response element (RRE) sequence. Rev binds to RRE in vitro with an apparent dissociation constant of 1 to 3 nM as determined by filter binding, gel mobility shift assays, or an immunoprecipitation assay using a monoclonal antibody specific for the Rev C-terminus. Antisense RRE sequences are bound by Rev with a 20-fold lower affinity than wild-type RRE sequences. The Rev-RRE complex forms even in the presence of a 10,000-fold molar excess of 16S rRNA, whereas formation of the low affinity antisense RRE-Rev complex is efficiently blocked by addition of excess 16S rRNA. A approximately 33 nucleotide fragment is protected from ribonuclease T1 digestion by the binding of Rev to RRE RNA, suggesting that Rev binds with high affinity to only a restricted region of the RRE. This protected fragment is unable to rebind Rev protein but has been mapped to a 71 nucleotide long Rev binding domain sequence that overlaps the protected fragment.

Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.

tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Cloning and expression in E. coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin.

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasmin was constructed and expressed in Escherichia coli as a fusion with beta-galactosidase. The gene was designed with a recognition site for the plasma protease, Factor Xa, coded for immediately prior to the N-terminus of caltrin. The beta-galactosidase-caltrin fusion protein was cleaved with Factor Xa to give caltrin, which was identified by its size on SDS-PAGE, its ability to react with an antiserum raised to the N-terminal nonapeptide of caltrin and its N-terminal amino acid sequence. After partial purification, synthetic caltrin was found to be active in an assay involving inhibition of growth of E.coli.