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At least 1,081 records · Page 60Linked to original sources
PubMed1984

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.

Bacterial Proteins↗
PubMed1985

DNA packaging initiation of Salmonella bacteriophage P22: determination of cut sites within the DNA sequence coding for gene 3.

DNA packaging of Salmonella phage P22 starts at a defined site on a concatemer of P22 genomes. The molecular ends formed at the packaging initiation site (pac) map within a region of ca. 120 base pairs and may contain any of the four nucleotides at their 5' end. The determination of the positions of the cuts within the sequence demonstrates a characteristic distribution of cut sites which apparently cannot be attributed to the sequence organization of the involved regions. Symmetric elements of the sequence might serve as signals for a recognition event(s) at pac in a separate process preceding the cutting reaction. The region of packaging initiation is located within the sequence coding for gene 3. The 3 protein is responsible for the site specificity of this process. We find no significant homology to Nu1 protein, which appears to have an analogous or similar function in the DNA maturation of Escherichia coli phage lambda.

Base Sequence↗
PubMed1987

Replication and packaging of choleraphage phi 149 DNA.

The intercellular replication of the circularly permuted DNA of choleraphage phi 149 involves a concatemeric DNA structure with a size equivalent to six genome lengths. The synthesis of both monomeric and concatemeric DNAs during replication of phi 149 occurred in the cytoplasm. The concatemers served as the substrate for the synthesis of mature phage DNA, which was eventually packaged by a headful mechanism starting from a unique pac site in the concatemeric DNA. Packaging of DNA into phage heads involved binding of concatemeric DNA to the cell membrane. A scheme involving sequential packaging of five headfuls proceeding in the counterclockwise direction from the pac site is proposed. After infection under high-phosphate conditions, the concatemeric DNA intermediates were not formed, although synthesis of monomeric molecules was unaffected.

Bacterial Proteins↗
PubMed1987

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.

Gene Products, gag↗
PubMed1989

Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it.

The single-stranded DNA of filamentous phages (f1, fd, M13, Ike) contains a region that can fold into a hairpin structure that serves to earmark the DNA for encapsidation. Second-site suppressor mutants of f1 that can compensate for deletion of this packaging signal have been isolated and characterized. The mutations lie in three genes, two that encode virion proteins located at the end of the particle that is first to emerge from the cell, the end at which the packaging signal is located, and the third in a gene whose product is required for assembly but which is not itself a part of the virion. Analysis of base substitution and deletion mutations in the packaging signal suggests that both structural and sequence elements are important to its proper function.

Bacteriophages↗
PubMed1992

Functional study of hepatitis delta virus large antigen in packaging and replication inhibition: role of the amino-terminal leucine zipper.

The large hepatitis delta antigen (HDAg) has been found to be essential for the assembly of the hepatitis delta virion. Furthermore, in a cotransfection experiment, the large HDAg itself, without the hepatitis delta virus (HDV) genome and small HDAg, could be packaged into hepatitis B surface antigen (HBsAg) particles. By deletion analysis, it was shown that the amino-terminal leucine zipper domain was dispensable for packaging. The large HDAg could also help in copackaging of the small HDAg into HBsAg particles without the need for HDV RNA. This process was probably mediated through direct interaction of the two HDAgs as a mutated large HDAg whose leucine zipper domain was deleted such that it could not help in copackaging of the small HDAg. This mutated large HDAg did not suppress HDV replication, suggesting that this effect is probably also via protein interaction. These results indicated that functional domains of the large HDAg responsible for packaging with HBsAg particles and for the trans-negative effect on HDV replication can be separated.

Antigens, Viral↗
PubMed1992

Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA). The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message. The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments. The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand. We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments. Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments. Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments. The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.

Bacteriophages↗
PubMed1994

Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis.

Hepatitis B viruses encode a polymerase (P) protein with key roles in both reverse transcription and genomic RNA encapsidation. Genetic analysis of cis-acting signals required for viral replication implicates an RNA stem-loop structure in both RNA packaging and the initiation of reverse transcription, a process in which P protein also serves as the primer. We now show that duck hepatitis B virus (DHBV) polymerase binds specifically and with high affinity to this RNA stem-loop structure. Mutational analysis indicates that all mutations in the RNA target that inhibit the P protein-RNA interaction inhibit both in vivo RNA packaging and in vitro DNA priming to comparable extents. However, certain mutations in the loop region of the RNA have minimal impact on P protein-RNA binding but are nonetheless severely defective for packaging and DNA synthesis. Thus, P protein-RNA complex formation is necessary but not sufficient to initiate these activities. In addition, examination of RNA binding by truncated P proteins indicates that the C terminus of the polymerase, although required for RNA encapsidation in vivo, is dispensable for RNA binding and DNA priming.

Base Sequence↗
PubMed1995

cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.

Animals↗
PubMed1995

Symmetric-strand packaging of recombinant parvovirus LuIII genomes that retain only the terminal regions.

LuIII is an autonomous parvovirus which encapsidates either strand of its genome with similar efficiency in NB324K cells. Two parvoviruses closely related to LuIII, minute virus of mice (MVM) and H-1 virus, encapsidate primarily the minus strand of their genome when grown in the same cell type. It has been postulated that an AT-rich region unique to LuIII is responsible for symmetric encapsidation of plus- and minus-strand genomes by LuIII. To address this hypothesis, recombinant LuIII-luciferase genomes containing or lacking the AT-rich sequence (AT) were packaged into LuIII virions. Hybridization of strand-specific probes to DNA from these virions revealed that either strand of the genome was packaged regardless of the presence of AT. In addition, encapsidation of both strands of the AT+ LuIII-luciferase genome into MVM and H-1 virions was observed, suggesting that MVM and H-1 viral proteins are not responsible for the minus-strand packaging bias of these two viruses. Alignment of the published LuIII and MVMp sequences shows that AT exists as an insertion into an element that, in MVM, binds cellular proteins. We suggest that in LuIII, AT disrupts binding of these cellular proteins, allowing encapsidation of either strand.

Base Sequence↗
PubMed1996

A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis.

In retroviruses, the genomic RNA is in the form of a 60S-70S complex composed of two identical genome-length RNA molecules tightly associated through numerous interactions. A major interaction, called the dimer linkage structure, has been found near the RNA 5' end and is probably involved in the control of translation, packaging, and recombination during proviral DNA synthesis. Recently, a small sequence corresponding to a stem-loop structure located in the 5' leader of human immunodeficiency virus type 1 (HIV-1) RNA was found to be required for the initiation of HIV-1 RNA dimerization in vitro and named the dimerization initiation site (E. Skripkin, J.-C. Paillart, R. Marquet, B. Ehresmann, and C. Ehresmann, Proc. Natl. Acad. Sci. USA 91: 4945-4949, 1994). To investigate the possible role of this 5' stem-loop in HIV-1 virion formation and infectivity, four mutant viruses were generated and analyzed in vivo. Results show that deletion of the stem-loop structure reduces infectivity by a factor of 10(3) whereas loop substitutions cause a decrease of 10- to 100-fold. The level of genomic RNA packaging was found to be decreased fivefold in mutants virions containing the stem-loop deletion and only twofold in the loop-substituted virions. Surprisingly, the second DNA strand transfer during reverse transcription was found to be severely impaired upon stem-loop deletion. Taken together, these results indicate that the stem-loop structure called the dimerization initiation site is a cis element acting on both genomic RNA packaging and synthesis of proviral DNA.

Animals↗
PubMed1996

Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

Animals↗
PubMed1997

In vitro selection of packaging sites in a double-stranded RNA virus.

The Saccharomyces cerevisiae double-stranded RNA virus ScVL1 recognizes a small sequence in the viral plus strand for both packaging and replication. Viral particles will bind to this viral binding sequence (VBS) with high affinity in vitro. An in vitro selection procedure has been used to optimize binding, and the sequences isolated have been analyzed for packaging and replication in vivo. The selected sequence consists of a stem with a bulged A residue topped by a loop of several bases. Four residues of the 18 bases are absolutely conserved for tight binding. These all fall in regions that appear to be single stranded. Eight more residues have preferred identities, and six of these are in the stem. The VBS is similar to the R17 bacteriophage coat protein binding site. Packaging and replication require tight binding to viral particles.

Binding Sites↗
PubMed1997

Gene transfer to human cells using retrovirus vectors produced by a new polytropic packaging cell line.

We report here the construction of a new packaging cell line, called MPAC, that packages defective retroviral vectors in viral particles with envelope proteins derived from a Moloney mink cell focus-inducing (MCF) polytropic virus. We characterized the tropism of MPAC-packaged retroviral vectors and show that some human cell lines can be infected with these vectors while others cannot. In addition, we show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication.

3T3 Cells↗
PubMed1998

Nucleocapsid and matrix protein contributions to selective human immunodeficiency virus type 1 genomic RNA packaging.

The nucleocapsid protein (NC) of retroviruses plays a major role in genomic RNA packaging, and some evidence has implicated the matrix protein (MA) of certain retroviruses in viral RNA binding. To further investigate the role of NC in the selective recognition of genomic viral RNA and to address the potential contribution of MA in this process, we constructed chimeric and deletion human immunodeficiency virus type 1 (HIV-1) mutants that alter the NC or MA protein. Both HIV and mouse mammary tumor virus (MMTV) NC proteins have two zinc-binding domains and similar basic amino acid compositions but differ substantially in total length, amino acid sequence, and spacing of the zinc-binding motifs. When the entire NC coding sequence of HIV was replaced with the MMTV NC coding sequence, we found that the HIV genome was incorporated into virions at 50% of wild-type levels. Viruses produced from chimeric HIV genomes with complete NC replacements, or with the two NC zinc-binding domains replaced with MMTV sequences, preferentially incorporated HIV genomes when both HIV and MMTV genomes were simultaneously present in the cell. Viruses produced from chimeric MMTV genomes in which the MMTV NC had been replaced with HIV NC preferentially incorporated MMTV genomes when both HIV and MMTV genomes were simultaneously present in the cell. In contrast, viruses produced from chimeric HIV genomes containing the Moloney NC, which contains a single zinc-binding motif, were previously shown to preferentially incorporate Moloney genomic RNA. Taken together, these results indicate that an NC protein with two zinc-binding motifs is required for specific HIV RNA packaging and that the amino acid context of these motifs, while contributing to the process, is less crucial for specificity. The data also suggest that HIV NC may not be the exclusive determinant of RNA selectivity. Analysis of an HIV MA mutant revealed that specific RNA packaging does not require MA protein.

Amino Acid Sequence↗
PubMed1998

Retrotransposition of nonviral RNAs in an avian packaging cell line.

Retroviruses produced from the quail packaging cell line SE21Q1b predominantly contain cellular RNAs instead of viral RNAs. These RNAs can be reverse transcribed and integrated into the genomes of newly infected cells and are thereafter referred to as newly formed retrogenes. We investigated whether retrogene formation can occur within SE21Q1b cells themselves and whether this occurs intracellularly or via extracellular reinfection. By using packaging cell line mutants derived from the SE21Q1b provirus and selectable reporter constructs, we found that the process requires envelope glycoproteins and a retroviral packaging signal. Our results suggest that extracellular reinfection is the primary route of retrotransposition of nonviral RNAs.

Animals↗
PubMed1998

Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors.

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10(4) vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.

Cell Line, Transformed↗
PubMed1999

Functional coupling between replication and packaging of poliovirus replicon RNA.

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

Base Sequence↗