Biomedical subjects

M L Linial

Publications and source records attributed to M L Linial.

At least 37 records · Page 2Linked to original sources

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

A minimal avian retroviral packaging sequence has a complex structure.

We have defined a 160-nucleotide region, Mpsi, from the 5' leader region of the Rous sarcoma virus genome that is sufficient to direct the packaging of a heterologous RNA. Mpsi contains the putative O3 stem structure that has previously been shown, and that has been confirmed in this study, to be important for the efficient packaging of avian leukosis-sarcoma virus RNA. Analyses of several O3 stem mutants revealed that other regions within Mpsi can interfere with the proper folding of altered sequences which are predicted to form a wild-type O3 stem.

Avian Sarcoma Viruses↗

PubMed1997

Tail-to-head arrangement of a partial chicken glyceraldehyde-3-phosphate dehydrogenase processed pseudogene.

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5' region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization.

Animals↗

PubMed1996

Viral Myc oncoproteins in infected fibroblasts down-modulate thrombospondin-1, a possible tumor suppressor gene.

We are interested in identifying the transcriptional targets of the Myc oncoproteins. To this end, we have fused Myc of the MC29 retrovirus with the rat glucocorticoid receptor. This chimeric protein requires dexamethasone to undergo nuclear translocation and achieve an active conformation. We employed a differential hybridization approach to identify mRNAs that are induced or repressed in infected avian fibroblasts in response to dexamethasone. This screen yielded one mRNA underrepresented in the dexamethasone-treated cells. In Myc-transformed cell clones, its level decreases 6-fold as early as 4 h and more than 30-fold after 32 h of exposure to the hormone. This mRNA was also down-regulated by recombinant Myc retroviruses in rodent fibroblasts, including those refractory to transformation. Sequence analysis revealed that it is homologous to the 3' untranslated regions of the mammalian thrombospondin-1 genes. Using an anti-thrombospondin antibody, we confirmed that rodent cells overexpressing Myc produce very small amounts of this protein. Also, they do not support efficient expression of a reporter gene driven by the thrombospondin-1 promoter. Thus, thrombospondin-1 is a bona fide target of Myc. Moreover, its silencing might pertain to the transforming activity of Myc, since in several systems thrombospondin-1 exhibits tumor suppressor properties, presumably due to its negative effect on neovascularization.

Amino Acid Sequence↗

PubMed1996

Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses.

Human foamy virus (HFV) is the prototype of the Spumavirus genus of Retroviridae. In all other retroviruses, the pol gene products, including reverse transcriptase, are synthesized as Gag-Pol fusion proteins and are cleaved to functional enzymes during viral budding or release. In contrast, the Pol protein of HFV is translated from a spliced messenger RNA and lacks Gag domains. Infectious HFV particles contain double-stranded DNA similar in size to full-length provirus, suggesting that reverse transcription has taken place in viral particles before new rounds of infection, reminiscent of hepadnaviruses. These data suggest that foamy viruses possess a replication pathway containing features of both retroviruses and hepadnaviruses but distinct from both.

Amino Acid Sequence↗

PubMed1996

Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence.

We developed an in vitro binding assay to study the specific interaction between human immunodeficiency virus type 1 (HIV-1) RNA and the Gag polyprotein. Binding of the in vitro-expressed protein to in vitro-transcribed RNA was determined by altered migration of the protein in polyacrylamide gels. We found that a Gag precursor lacking the matrix domain bound specifically to HIV-1 RNA, while deletion of both matrix and capsid domains diminished the specificity of binding. Among several regions of HIV-1 RNA tested, strongest binding was seen with the 5'-most 261 nucleotides, while antisense RNA from the same region did not bind.

Animals↗

PubMed1996

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains.

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein.

Amino Acid Sequence↗

PubMed1996

Productive persistent infection of hematopoietic cells by human foamy virus.

Human foamy virus can establish persistent infections in human hematopoietic cell lines, such as H92.1.7 (erythroblastoid cells), Jurkat (CD4+ T cells), and U937 (myeloid-monocytic cells). The infection is characterized by constant production of infectious viruses (for > 2 1/2 years) with no cytopathic effects on the host cells. Electron microscopy of the infected cells showed a viral morphology similar to that observed for particles produced after acute infection. We have detected, in addition to the full-length form of bel1, a previously described deletion in the bel1 gene of the proviral DNA in these cells. RNA containing this 301-bp deletion, which mapped to the splice donor and acceptor sites of the intron of the bet gene, was also found in encapsidated virion RNA. However, the presence of this defective provirus harboring the deletion in bel1 does not prevent productive persistence in these chronically infected cells, since the virus titer does not decrease during cultivation.

Base Sequence↗

PubMed1995

v-Myc is invariably required to sustain rapid proliferation of infected cells but in stable cell lines becomes dispensable for other traits of the transformed phenotype.

The v-myc-containing retrovirus MC29 induces neoplastic transformation of avian embryo cells. To determine which traits of the transformed phenotype are directly controlled by v-Myc, we engineered a conditional MC29 mutant (GRIM) expressing v-Myc as a fusion protein with the glucocorticoid receptor and the retroviral Gag polyprotein. Only in the presence of glucocorticoids such as dexamethasone is GRIM capable of transforming embryo cells, from which six stable GRIM-lines have been derived. Although their survival in culture no longer requires functional v-Myc, hormone deprivation causes all six GRIM clones as well as acutely infected fibroblast cultures to either withdraw from cell cycle completely or to grow much more slowly and to much lower densities. However, removal of dexamethasone does not allow GRIM-transformed mass cultures and most of the clones to revert to normal shapes or to reconstruct actin cables. Furthermore, most clones do not require the hormone sustain anchorage-independent growth. We propose that certain secondary events have let the GRIM-clones sustain immortality, transformed morphology, and anchorage-independent growth independently of v-Myc. None of these events, however, has obliterated the requirement for v-Myc in cell division control. We thus conclude that enhanced proliferation is the primary effect of v-Myc expression.

Actin Cytoskeleton↗

PubMed1995

Inhibition of wild-type HIV-1 virus production by a matrix deficient Gag mutant.

Previous studies have shown that certain HIV-1 Gag mutants can interfere with the production of infectious HIV-1 when coexpressed with wild-type virus. In this paper, we studied two mutants of HIV-1 for their ability to interfere with the production of wild-type virus. Both mutants lack the entire matrix domain of gag and either lack [myr(-)MA(-)] or contain [myr(+)MA(-)] an amino-terminal myristate (myr) addition sequence at the beginning of the capsid domain. Previously we have demonstrated that expression of both mutant constructs leads to assembly and release of mutant viruses, although only myr(+)MA(-) particles are released efficiently. Particles produced by both matrix-deficient mutants are noninfectious and poorly incorporate and/or retain viral envelope glycoproteins. In this study, we further show that expression of myr(+)MA(-), but not myr(-)MA(-) interferes with wild-type HIV-1 virus production in transient expression assays. Our data suggest that wild-type and myristylated MA(-) Gag protein interacts at some point during assembly and that Gag myristylation has a greater effect on the assembly pathway than the matrix domain.

Amino Acid Sequence↗

PubMed1995

The human foamy virus internal promoter is required for efficient gene expression and infectivity.

The human foamy or spumaretrovirus (HFV) is a complex retrovirus that codes for the three retroviral genes gag, pol, and env and the regulatory and accessory bel genes. A particular feature of HFV gene expression was recently described: not only does the HFV provirus contain the classical retroviral long terminal repeat promoter, a second functionally active promoter is present in the env gene upstream of the bel genes (M. Löchelt, W. Muranyi, and R. M. Flügel, 1993, Proc. Natl. Acad. Sci. USA 90, 7317-7321). Both, the HFV long terminal repeat promoter I and internal promoter II depend upon the HFV transcriptional transactivator Bel 1 for efficient gene expression. The internal promoter directs the synthesis of functionally active Bel 1 transactivator and Bet proteins that are expressed early after HFV infection. In this report, it is shown that mutation of the promoter II TATA box resulted in HFV proviral clones with a reduction in infectivity by a factor of approximately 100. Gene expression by promoter II TATA box mutant HFV proviruses was reduced. HFV proviruses with the mutated promoter II TATA box used cryptic start sites of transcription upstream of the original promoter II TATA box, resulting in an inefficient and less accurate transcriptional initiation. The reduced HFV structural gene expression by the mutated HFV proviruses was relieved by providing Bel 1 protein in trans. This demonstrates that HFV promoter II-directed Bel 1 expression is important for producing the high levels of Bel 1 that increases virus replication.

Animals↗

PubMed1995

The packaging phenotype of the SE21Q1b provirus is related to high proviral expression and not trans-acting factors.

The avian packaging cell line SE21Q1b produces particles which encapsidate cellular RNAs. Such RNAs can be reverse transcribed by endogenous polymerase and integrated into the genomes of newly infected cells (M. Linial, Cell 49:93-102, 1987). Genomic RNA is not packaged because the packaging (psi) region of the provirus is deleted. The provirus also lacks the negative-strand primer binding site, which prevents efficient reverse transcription of randomly packaged genomic RNA. Previous work from our laboratory suggested that the trans-acting defect which allows packaging of cellular mRNA mapped to the provirus but did not map to the nucleocapsid region of the gag gene (D.J. Anderson, P. Lee, K. L. Levine, J. Sang, S. A. Shah, O. O. Yang, P. R. Shank, and M. L. Linial, J. Virol. 66:204-216, 1992). We have found, using proviral recombinants between SE21Q1b and wild-type Rous sarcoma virus, that packaging of cellular RNAs does not map to the gag gene. Rather, the propensity of SE21Q1b particles to package cellular mRNA is a function of the high level of particle production in these cells and not of any specific viral structural proteins.

Amino Acid Sequence↗

PubMed1995

Modification of retroviral RNA by double-stranded RNA adenosine deaminase.

In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions.

Adenosine↗

PubMed1994

Efficient particle formation can occur if the matrix domain of human immunodeficiency virus type 1 Gag is substituted by a myristylation signal.

Lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), assemble at and bud through the cytoplasmic membrane. Both the matrix (MA) domain of Gag and its amino-terminal myristylation have been implicated in these processes. We have created HIV-1 proviruses lacking the entire matrix domain of gag which either lack or contain an amino-terminal myristate addition sequence at the beginning of the capsid domain. Myristate- and matrix-deficient [myr(-)MA(-)] viruses produced after transient transfection are still able to assemble into particles, although the majority do not form at the plasma membrane or bud efficiently. Myristylation of the amino terminus of the truncated Gag precursor permits a much more efficient release of the mutant virions. While myr(-)MA(-) particles were inefficient in proteolytic processing of the Gag precursor, myristylation enabled efficient proteolysis of the mutant Gag. All matrix-deficient viruses are noninfectious. Particles produced by matrix-deficient mutants contain low levels of glycoproteins, indicating the importance of matrix in either incorporation or stable retention of Env. Since matrix-deficient viruses contain a normal complement of viral genomic RNA, a role for MA in genomic incorporation can be excluded. Contrary to previous reports, the HIV-1 genome does not require sequences between the 5' splice donor site and the gag start codon for efficient packaging.

Base Sequence↗

PubMed1993

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.

Animals↗

PubMed1993

Analysis of the role of the bel and bet open reading frames of human foamy virus by using a new quantitative assay.

We have constructed a BHK-21-derived indicator cell line containing a single integrated copy of the beta-galactosidase (beta-Gal) gene under control of the human foamy virus (HFV) long terminal repeat promoter (from -533 to +20). These foamy virus-activated beta-Gal expression (FAB) cells can be used in a quantitative assay to measure the infectious titer of HFV. Our results show that the FAB assay is 50 times more sensitive than determination of the virus titer by the end-point dilution method. Using the FAB assay, we have found that HFV can productively replicate in several erythroblastoid cell lines as well as in the Jurkat T-cell line. We have also examined the roles of bel2, bet, and bel3 in viral replication by constructing proviral HFV clones in which the reading frame of Bel2, Bet, or Bel3 is disrupted by placement of translation stop codons. Analysis of these mutants reveals that while the bel3 gene is not required for viral replication in vitro, mutations in the bel2 or bet gene decrease cell-free viral transmission approximately 10-fold.

Base Sequence↗

PubMed1993

Characterization of a unique retroviral recombinant containing 7S L sequences.

Using a recently described system to generate recombinants between avian leukosis viruses (ALV) and cellular neo mRNA (A.M. Hajjar and M.L. Linial, J. Virol. 67:3845-3853, 1993), we isolated a recombinant containing 7S L sequences. Analysis of this recombinant revealed that it most likely arose during reverse transcription of three copackaged RNAs: 7S L RNA, neo RNA, and ALV genomic RNA. Reverse transcription appears to have initiated on the 7S L RNA. A model for the generation of this recombinant is described.

Animals↗

PubMed1993

Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection.

The avian retrovirus FH3, which encodes a Gag-Myc fusion protein, transforms chicken macrophages but not fibroblasts. However, passage of FH3 viral stock in fibroblasts leads to emergence of a virus capable of fibroblast transformation. This virus has not acquired myc mutations; instead, it carries internal gag deletions which confer the ability to transform fibroblasts. We now demonstrate that this and similar deletion variants emerge repeatedly during selection. Sequence analysis reveals direct repeats at or near deletion junctions, suggesting that errors during reverse transcription may be involved in genesis of these viruses, which are then positively selected in fibroblast culture. By using the polymerase chain reaction, we found that such variants preexisted in original stocks even before selection, although they could not be detected by focus assay.

Animals↗