High-frequency cotransfer of the transformed phenotype and a tumor-specific transplantation antigen by DNA from the 3-methylcholanthrene-induced Meth A sarcoma of BALB/c mice.

Hopkins, Nancy; Besmer, Peter; DeLeo, Albert B.; Law, Lloyd W.

doi:10.1073/pnas.78.12.7555

Cited by 50 publications

(15 citation statements)

References 18 publications

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“…Virus stocks of NL4-3, ␣-helix 2 mutants, and VSV-G pseudotypes were obtained by transfecting HeLa cells using the calcium phosphate precipitation method (14,16). Transfected cell supernatants were harvested, filtered (0.45-m-pore-size filter), normalized for RT activity, and used in infections as described below.…”

Section: Methodsmentioning

confidence: 99%

Genetic Evidence for an Interaction between Human Immunodeficiency Virus Type 1 Matrix and α-Helix 2 of the gp41 Cytoplasmic Tail

Murakami

Freed

2000

J Virol

198

216

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The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (␣-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of ␣-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.Retroviral envelope (Env) glycoproteins are synthesized in the endoplasmic reticulum as precursor proteins that are proteolytically cleaved by a cellular protease during their transport to the cell surface (11,39). Cleavage of the Env precursor generates the two components of the mature Env glycoprotein complex: the surface (SU) and transmembrane (TM) Env glycoprotein subunits. In the case of human immunodeficiency virus type 1 (HIV-1), the precursor, SU, and TM Env glycoproteins have been designated gp160, gp120, and gp41, respectively. After reaching the plasma membrane, the Env complex is incorporated into budding virions by a process whose details remain to be elucidated.A striking feature of lentiviruses is that they encode TM Env glycoproteins with cytoplasmic tails (CTs) that are much longer than those of other retroviruses. The HIV-1 and HIV-2 TM CTs, for example, are generally around 150 residues in length, more than five times longer than those of the avian and murine oncoretroviruses. Previous studies have observed diverse effects of HIV-1 gp41 CT mutations, including defective Env incorporation (6, 9, 46), impaired virus infectivity (6, 9, 13), reduced gp160 processing and Env stability (13), disrupted basolateral targeting of virus release (2, 25), and slower Env internalization from the cell surface (3, 33, 36). The structure of the gp41 CT has not been determined; however, several regions within the tail are likely to adopt a helical folding (8,40). One of these, termed ␣-helix 1, is located at the C terminus of gp41; another, referred to as ␣-helix 2, is located near the center of the CT (9). These helical motifs have also been referred to as lentivirus lytic peptides due to their ability to associate with and disrupt lipid bilayers (20,28,29,38). The periodic spacing of Leu residues in ␣-helix 2 suggests that it may form a Leu zipper (23).Mixed results have been reported concerning whether or not the incorpora...

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Section: Methodsmentioning

confidence: 99%

Genetic Evidence for an Interaction between Human Immunodeficiency Virus Type 1 Matrix and α-Helix 2 of the gp41 Cytoplasmic Tail

Murakami

Freed

2000

J Virol

198

216

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“…Virus stocks of NL4-3, NL(AD8), CTdel-144-2 mutant derivatives, and VSV-G pseudotypes were obtained by transfecting HeLa cells by using the calcium phosphate precipitation method (30,31). Pseudotyped virus stocks of pNLuc were prepared by cotransfection of 293T cells with HIV-1 Env expression vectors as described (25).…”

Section: Methodsmentioning

confidence: 99%

The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Murakami

Freed

2000

Proc. Natl. Acad. Sci. U.S.A.

232

280

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Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a highlevel, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions. The HIV-1 envelope (Env) glycoprotein is synthesized as a precursor, gp160, that is proteolytically cleaved by a cellular protease during transport to the cell surface (1, 2). Cleavage of gp160 produces the two components of the mature Env glycoprotein complex: the surface Env glycoprotein gp120 and the transmembrane Env glycoprotein gp41. After its arrival at the plasma membrane, the gp120͞gp41 complex is incorporated into nascent, budding virus particles by a mechanism that remains poorly understood.Lentiviruses, including HIV-1, encode transmembrane glycoproteins with cytoplasmic tails (CTs) that are quite long compared with those of other retroviruses. For example, the CT of the HIV-1 and HIV-2 transmembrane glycoproteins are generally around 150 residues in length; in contrast, those of the avian and murine oncoretroviruses are typically 20-30 aa long. Although the role of the long lentiviral Env CT has been the focus of a number of studies, no clear consensus has emerged regarding the function of this unique domain. Some studies observed that deletions of more than 19 (3) or 43 (4) residues from the gp41 C terminus blocked virus infectivity; this phenotype was attributed in part to reduced Env incorporation into virions. Another study (5) observed that the gp41 CT played no major role in Env incorporation, but was required for virus infectivity. Wilk et al. (6) reported that a mutant lacking essentially all of the gp41 CT efficiently estab...

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“…DNAs ofa variety ofneoplasms from several different vertebrate species, including man, induce transformation of NIH 3T3 cells with high efficiencies, indicating that carcinogenesis can involve dominant genetic alterations resulting in the activation of cellular transforming genes (3)(4)(5)(6)(7)(8)(9)(10). Analysis of the transforming genes activated in neoplasms has suggested that different transforming genes are activated in different types ofneoplasms, but the same or closely related transforming genes are activated in independent neoplasms of the same type of differentiated cells (6-8, 10, 11).…”

mentioning

confidence: 99%

Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses.

Der¹,

Krontiris²,

Cooper³

1982

Proc. Natl. Acad. Sci. U.S.A.

671

210

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Blot hybridization analysis indicated that NIH 3T3 mouse cells transformed by high molecular weight DNAs of a human bladder and a human lung carcinoma cell line contained new sequences homologous, respectively, to the transforming genes of Harvey (rasH) and Kirsten (rasK) sarcoma viruses. The unique ras sequences were present in multiple independent NIH cell lines transformed in both primary and secondary transfection assays and corresponded to ras sequences normally present in human DNAs. The ras gene product was expressed in NIH cells transformed by bladder carcinoma DNAs and in the human bladder carcinoma cell lines at levels 2-to 4-fold greater than the level observed in nontransformed NIH 3T3 cells. These results indicate that the transforming genes of these human tumor cell lines are the cellular homologs of two retroviral transforming genes.Transfection of cellular DNAs has demonstrated that both normal and transformed cells contain genes capable of inducing oncogenic transformation (1, 2). DNAs ofa variety ofneoplasms from several different vertebrate species, including man, induce transformation of NIH 3T3 cells with high efficiencies, indicating that carcinogenesis can involve dominant genetic alterations resulting in the activation of cellular transforming genes (3-10). Analysis of the transforming genes activated in neoplasms has suggested that different transforming genes are activated in different types ofneoplasms, but the same or closely related transforming genes are activated in independent neoplasms of the same type of differentiated cells (6-8, 10, 11).One class of potential cellular transforming genes is composed of the cellular homologs of the transforming genes of highly oncogenic retroviruses. These viruses contain specific genes that are responsible for transformation and that are homologous to genes ofnormal cells (12). At least a dozen different viral transforming genes have been identified, each of which has a cellular homolog (13). These cellular sequences are highly conserved in vertebrate evolution, but their function in both normal and neoplastic cells has been unclear.In the present study, we have investigated the possible relationship between the cellular homologs of retroviral transforming genes and the transforming genes detected by transfection of tumor DNAs. We report here that the transforming genes of human bladder and lung carcinoma cell lines are homologous to the transforming genes of Harvey and Kirsten sarcoma viruses. with 50 ,ul of staphylococcal protein A-Sepharose (50% suspension) that had been precoated with rabbit anti-rat immunoglobulin G. The protein A-Sepharose was removed by centrifugation and lysates were incubated for 1.5 hr at 40C with rat monoclonal antibody 259 against Harvey sarcoma virus p21 (generously provided by E. M. Scolnick). This monoclonal antibody recognizes broadly reactive p21 determinants which are present on cellencoded, rasH-encoded, and rasK-encoded gene products (M. MATERIALS AND METHODS CellFurth and E. M. Scolnick, personal c...

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High-frequency cotransfer of the transformed phenotype and a tumor-specific transplantation antigen by DNA from the 3-methylcholanthrene-induced Meth A sarcoma of BALB/c mice.

Cited by 50 publications

References 18 publications

Genetic Evidence for an Interaction between Human Immunodeficiency Virus Type 1 Matrix and α-Helix 2 of the gp41 Cytoplasmic Tail

Genetic Evidence for an Interaction between Human Immunodeficiency Virus Type 1 Matrix and α-Helix 2 of the gp41 Cytoplasmic Tail

The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses.

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