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M Emerman

Publications and source records attributed to M Emerman.

54 records · Page 3Linked to original sources
PubMed1992

Spleen necrosis virus, an avian immunosuppressive retrovirus, shares a receptor with the type D simian retroviruses.

The reticuloendotheliosis viruses (REV) are a family of highly related retroviruses isolated from gallinaceous birds. On the basis of sequence comparison and overall genome organization, these viruses are more similar to the mammalian type C retroviruses than to the avian sarcoma/leukemia viruses. The envelope of a member of the REV family, spleen necrosis virus (SNV), is about 50% identical in amino acid sequence to the envelope of the type D simian retroviruses. Although SNV does not productively infect primate or murine cells, the receptor for SNV is present on a variety of human and murine cells. Moreover, interference assays show that the receptor for SNV is the same as the receptor for the type D simian retroviruses. We propose that adaptation of a mammalian type C virus to an avian host provided the REV progenitor.

Amino Acid Sequence↗
PubMed1991

Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI.

The construction and preliminary biological characterization of three molecular clones of human immunodeficiency virus type 1 (HIV-1) are reported: HIV-1LAI from a French man with AIDS, HIV-1MAL from a Zairian boy with ARC, and HIV-1ELI from a Zairian woman with AIDS. All three sequences were found to code for infectious viruses. Both the host range and the kinetics of infection in CD4+ cells were different for the three viruses. Virus derived from each molecular clone was infectious on peripheral blood mononuclear cells (PBMC), although LAI and ELI displayed more rapid growth kinetics than MAL. The viruses had different tropisms and growth kinetics in six cell lines. LAI was infectious in all of the cell lines and produced high levels of reverse transcriptase activity. MAL and ELI had more restricted tropisms: MAL could only replicate on SupT1, whereas ELI grew on Jurkat and MT-4, was delayed on CEM and H9, and was unable to infect U937 cells. In addition, we observed that both the replicative capacity and the cell tropism of viruses could change after passage through some established cell lines. These results suggest that the genotypes of some viruses in vitro are not stable and that selection for growth can cause the fairly rapid appearance of variants with increased growth potential.

AIDS-Related Complex↗
PubMed1990

Activation of a beta-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells.

A quantitative bioassay for human immunodeficiency viruses has been developed on the basis of the ability of the tat gene to transactivate the expression of an integrated beta-galactosidase gene in a HeLa-CD4+ cell line. Infection by a single virion of HIV-1 or HIV-2 corresponds to a unique blue syncytium or a cell cluster detected after fixation and addition of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (a beta-galactosidase substrate). The number of infected lymphoid cells in a culture (stimulated human peripheral blood lymphocytes and cell lines) can also be quantified by cell-to-cell transmission of HIV into the HeLa-CD4(+)-beta-galactosidase monolayer. Infections by simian immunodeficiency viruses are similarly detected. This assay has been used to determine the dose response of drugs, the half-life of HIV at 37 degrees C, and the appearance of infectious particles after virus infection.

Blotting, Southern↗
PubMed1989

Single amino-acid changes in HIV envelope affect viral tropism and receptor binding.

Infection by the human immunodeficiency virus (HIV) is initiated by the binding of its extracellular envelope glycoprotein, gp120, to the CD4 antigen on target cells. To map the residues of the HIV-1 glycoprotein that are critical for binding and to analyse the effects of binding on viral infectivity, we created 15 mutations in a region of gp120 that is important for binding to CD4 (refs 4,5). We find that substitution of a single amino acid (tryptophan at position 432) can abrogate CD4 binding and that virus carrying this mutation is non-infectious. By contrast, other amino-acid changes in the same region do not affect CD4 binding but restrict viral tropism: virions containing isoleucine substitutions at position 425 lose their ability to infect a monocyte cell line (U937 cells) but can still infect T-lymphocyte cell lines (CEM, SUP-T1) and activated human peripheral blood lymphocytes. These results indicate that cellular tropism of HIV can be influenced by a single amino-acid change in gp120.

HIV↗
PubMed1989

The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization.

By in situ hybridization analysis and immunoprecipitations following transfection of COS cells, we show that the Rev protein of the human immunodeficiency virus is necessary for envelope protein expression, which is correlated with the appearance in the cytoplasm of envelope-specific RNA. In the absence of cotransfection with a plasmid expressing Rev, envelope-specific RNA is retained in the nucleus. Several cis-acting sites in the envelope are involved, one of which is between nucleotides 7330 and 7735 and is required for the response to Rev. Other sequences (nucleotides 5797-7330 and 7735-7989) are involved in the apparent retention of the envelope-specific RNA in the nucleus in the absence of Rev and its response element. Because Rev affects the localization of envelope RNA both in the presence and in the absence of the normal splice sites on the RNA, the mechanism of Rev action is independent of splicing.

Cell Compartmentation↗
PubMed1989

VPX mutants of HIV-2 are infectious in established cell lines but display a severe defect in peripheral blood lymphocytes.

Nucleotide sequence comparison between HIV-1, HIV-2 and SIV has revealed the presence of an open reading frame (ORF) in the central region of the genomes of HIV-2 and SIV that has no counterpart in HIV-1. This new ORF, called vpx, is highly conserved between HIV-2ROD and SIVmac. Using anti-peptide sera to the predicted protein and site-directed mutagenesis, we show that mutations in the vpx ORF eliminate the synthesis of a 16 kd protein in HIV-2 infected cells, confirming that this protein is the product of this gene. Full-length clones of HIV-2 containing these mutations are infectious in two permanent T lymphocytic cell lines and two monocytic cell lines. In contrast, we show that loss of VPX function results in a severe defect in the productive infection of human peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings suggest that the VPX protein plays an important role in the in vivo life cycle of the HIV-2/SIV viruses.

Amino Acid Sequence↗
PubMed1989

The cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity.

A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection.

Codon↗
PubMed1989

Effects of mutations in hyperconserved regions of the extracellular glycoprotein of human immunodeficiency virus type 1 on receptor binding.

Sequence comparison of the human immunodeficiency virus type 1 and type 2 env genes revealed the presence of six linear regions in the extracellular glycoprotein that are highly conserved. To investigate the functional significance of these regions, we made short deletions in each and assayed the ability of the mutated proteins to bind CD4 antigen. Small deletions in four of the highly conserved regions drastically reduced receptor binding. Some deletions interfered with the maturation of the envelope glycoprotein, but maturation did not necessarily correlate with the ability to bind CD4 antigen.

Amino Acid Sequence↗
PubMed1987

The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1.

The recently described human immunodeficiency virus type 2 (HIV2) is significantly divergent in sequence from the more frequently isolated human immunodeficiency virus type 1 (HIV1). Both HIV1 and HIV2 encode a transactivator that is capable of strongly stimulating expression directed by the viral long terminal repeat (LTR). Here, we define the region of the HIV2 genome encoding the transactivator and show that the specificity of the transactivator differs from that of HIV1. By deletion analysis of the HIV2-LTR, we show that both HIV1 and HIV2 transactivators require sequences within 35 to 53 bp downstream of the start of transcription. However, in order to stimulate expression at full efficiency, the HIV2 transactivator further requires sequences unique to the HIV2-LTR between nucleotides +53 and +99. Hence, HIV2 poorly transactivates the LTR of HIV1, while two divergent isolates of HIV1 will efficiently transactivate the LTR of either HIV1 or HIV2. Nonetheless, in vivo competition between the transactivators of HIV1 and HIV2 suggests that they use a common mechanism.

Base Sequence↗
PubMed1987

Instability of large direct repeats in retrovirus vectors.

Retrovirus vectors were constructed with large (0.85- to 1.3-kilobase-pair) direct repeats in their genomes. Deletions involving the direct repeats occurred at a high frequency. Deletions occurred both when the direct repeats were in tandem and when they were separated by additional sequences. These deletions occurred during virus replication.

Base Sequence↗
PubMed1986

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.

Chromatin↗
PubMed1986

Quantitative analysis of gene suppression in integrated retrovirus vectors.

Previously, we described a retrovirus vector that contained two genes: a 5' gene under transcriptional control of the viral long terminal repeat and a 3' gene under transcriptional control of the herpes simplex virus thymidine kinase promoter. By using a biological assay, we found that expression of the 5' gene is suppressed when there was selection for the 3' gene and expression of the 3' gene is suppressed when there is selection for the 5' gene (M. Emerman and H. M. Temin, Cell 39:459-467, 1984). In the present study, we replaced the thymidine kinase promoter with stronger promoters, and we measured expression of the genes in the retrovirus vectors by enzyme activity and RNA analysis. We found that all of the vectors displayed gene suppression when analyzed biochemically, although not when analyzed biologically. The suppressed genes produced about 10 to 50% as much product as when they were selected. The amount of suppression depended on whether the suppressed gene was 5' or 3' to the selected gene and from which promoter the suppressed gene was transcribed. The amount of suppression correlated with a decrease in the amount of steady-state RNA transcribed from the suppressed gene's promoter.

Animals↗
PubMed1984

High-frequency deletion in recovered retrovirus vectors containing exogenous DNA with promoters.

We previously described infectious retrovirus vectors constructed from spleen necrosis virus which contain the herpes simplex virus thymidine kinase gene and the mouse alpha-globin gene (K. Shimotohno and H. M. Temin, Nature [London] 299:255-268, 1982). In the present study we report that when TK- chicken cells infected with a virus containing the mouse alpha-globin promoter and other 5' noncoding sequences in addition to the alpha-globin coding sequences were selected for thymidine kinase (TK) activity, all virus-producing TK+ cell clones shed virus with a deletion. These deletions were of different sizes and included the mouse alpha-globin coding sequences and the mouse alpha-globin transcriptional promoter. One of the deleted viruses was molecularly cloned. DNA sequencing showed that the deleted sequences are flanked by a short direct repeat. This deleted virus was also shown to have an advantage over the nondeleted parent both in multiplication and in its specific TK-transforming unit titer. In contrast to the results described above, TK+ cell clones established with viruses that contained only the coding sequences from the mouse alpha-globin gene did not delete and were stable over many cell passages. The implications of the high-frequency deletion of the viruses with internal promoters are discussed in terms of the evolution of retroviruses and the construction of retrovirus vectors.

Animals↗
PubMed1984

Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism.

We have constructed retrovirus vectors containing two genes, each with its own promoter, whose products can be selected in vivo. In cell clones harboring a single copy of this bicistronic provirus, we find that the 3' gene is usually suppressed when there is selection for expression of the 5' gene, and the 5' gene is usually suppressed when there is selection for expression of the 3' gene. We find that the suppression is epigenetic, reversible, and cis-acting, and is influenced by the strength or position of the different promoters. This type of gene suppression is novel for its action on a gene that can be either upstream or downstream to an adjacent active gene.

Animals↗
PubMed1982

Cleavage and cross-linking of proteins with osmium (VII) reagents.

The specific cleavage of proteins following reaction with osmium tetroxide is slowed down by tertiary amines which are known to be good ligands for osmium. Bidentate ligands are more effective than monodentate ligands. These same ligands also promote an intermolecular cross-linking reaction.

Cross-Linking Reagents↗
PubMedDate not supplied

Genome organization and transactivation of the human immunodeficiency virus type 2.

Analysis of the nucleotide sequence of the human retrovirus associated with AIDS in West Africa, HIV-2, shows that it is evolutionarily distant from the previously characterized HIV-1. We suggest that these viruses existed long before the current AIDS epidemics. Their biological properties are conserved in spite of limited sequence homology; this may help the determination of the structure-function relationships of the different viral elements.

Acquired Immunodeficiency Syndrome↗