Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-1 gag and pol gene products in stably transfected rodent cell lines

Moosmayer, Dieter; Reil, Heide; Ausmeier, Martina; Scharf, Jens‐Gerd; Hauser, Hansjörg; Jentsch, Klaus Dieter; Hunsmann, Gerhard

doi:10.1016/0042-6822(91)90134-w

Cited by 14 publications

(11 citation statements)

References 52 publications

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“…Upon infection with NL4-3(VSV-G) pseudotypes, the murine cells produced large amounts of Gag and Gag-Pol, but this accumulated in the cells in vesicular structures and was largely not released. The Gag proteins were inefficiently pro- cessed by the viral protease, probably as a result of their failure to assemble, consistent with earlier findings in transfected rodent cells (35). Importantly, the infected murine cells produced small amounts of virions, and these were as infectious on a per-particle basis as those from human cells, consistent with findings by Garber et al (18), who used transfected NIH 3T3 cells.…”

Section: Discussionsupporting

confidence: 90%

“…The presence of pr160 gag/pol at an abundance similar to that of GHOST.R5 suggested that the translational frameshifting event required for synthesis of the Gag-Pol polyprotein occurred in the murine cells at a frequency comparable to that in human cells, consistent with an earlier report by Moosmayer et al (35). Importantly, while the pr55 gag polyprotein was present at a similar abundance in the murine and human cells, fully processed CA was significantly reduced in abundance in the murine cells.…”

Section: Expression Of Hu-cd4 Hu-ccr5 and Hu-cyclin T1 In Murine Cesupporting

confidence: 85%

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A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells

Mariani

Rütter

Harris

et al. 2000

J Virol

145

175

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Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24 gag into the culture medium. A small amount of p24 gag was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.Several murine models have been developed for studies of AIDS pathogenesis. Mice transgenic for either the entire or partial human immunodeficiency virus type 1 (HIV-1) genome develop symptoms with similarities to AIDS. In one model, mice expressing HIV-1 Nef developed a wasting syndrome characterized by the loss of CD4 ϩ cells (21, 45). In another model, SCID mice were reconstituted with human peripheral blood lymphocytes or fetal thymus and liver and then inoculated with HIV-1 (37). These have been useful for studies on mechanisms of CD4 ϩ cell depletion and for evaluation of therapeutic strategies.Current murine models lack a central feature of HIV-1-induced pathogenesis: virus replication. Inoculation of mice or rodents such as rats, hamsters, and guinea pigs with high-titer HIV-1 does not result in detectable viremia, nor does the virus replicate in murine cells in culture (37) or infect human-CD4 (hu-CD4) transgenic mice (33). Low levels of virus replication have been detected in experimentally infected rabbits (13,15,20,26,43) and cotton rats (30), but this does not induce pathogenesis. Development of a system in which HIV-1 could replicate in mice would allow the investigation of features...

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Section: Discussionsupporting

confidence: 90%

Section: Expression Of Hu-cd4 Hu-ccr5 and Hu-cyclin T1 In Murine Cesupporting

confidence: 85%

A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells

Mariani

Rütter

Harris

et al. 2000

J Virol

145

175

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“…Nevertheless, with the possible exception of Chinese hamster ovary cells, none of the cell lines tested supports efficient virus replication. Although entry, integration, and expression of virus proteins occur efficiently, virion assembly and Gag polyprotein processing are severely impaired, resulting in markedly reduced levels of virus release and the failure of the virus to spread (1,9,11). The assembly defect is unlikely to be due to the inability of the human immunodeficiency virus type 1 (HIV-1) protease to function in murine cells, because processing is not required for assembly (4) and protease activity in murine cells has been demonstrated (6).…”

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confidence: 99%

Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Reed

Mariani

Sheppard

et al. 2002

J Virol

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Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.Murine cells expressing hu-CD4, CCR5, and hu-cyclin T1 support the early steps in the retrovirus replication cycle, including entry, reverse transcription, and integration. Following integration, sufficient quantities of viral mRNA transcripts are synthesized, and the cells accumulate abundant quantities of virion structural proteins. Nevertheless, with the possible exception of Chinese hamster ovary cells, none of the cell lines tested supports efficient virus replication. Although entry, integration, and expression of virus proteins occur efficiently, virion assembly and Gag polyprotein processing are severely impaired, resulting in markedly reduced levels of virus release and the failure of the virus to spread

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“…Changes in PRF efficiency alter those ratios, inhibiting virion morphogenesis (9). In particular, retroviruses appear to be very susceptible to such changes (4,21,33,37). Thus, elucidation of the molecular mechanisms underlying PRF can aid in the rational design of antiviral therapeutics (reviewed in reference 8).…”

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confidence: 99%

Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed −1 Ribosomal Frameshifting

Muldoon-Jacobs

Dinman

2006

Eukaryot Cell

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The ribosome-associated molecular chaperone complexes RAC (Ssz1p/Zuo1p) and Ssb1p/Ssb2p expose a link between protein folding and translation. Disruption of the conserved nascent peptide-associated complex results in cell growth and translation fidelity defects. To better understand the consequences of deletion of either RAC or Ssb1p/2p, experiments relating to cell growth and programmed ribosomal frameshifting (PRF) were assayed. Genetic analyses revealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resulted in specific inhibition of ؊1 PRF and defects in Killer virus maintenance, while no effects were observed on ؉1 PRF. These factors may provide a new set of targets to exploit against viruses that use ؊1 PRF. Quantitative measurements of growth profiles of isogenic wild-type and mutant cells showed that translational inhibitors exacerbate underlying growth defects in these mutants. Previous studies have identified ؊1 PRF signals in yeast chromosomal genes and have demonstrated an inverse relationship between ؊1 PRF efficiency and mRNA stability. Analysis of published DNA microarray experiments reveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcript levels are regulated independently of those of genes encoding ribosomal proteins. Thus, the findings presented here suggest that these trans-acting factors could be used by cells to posttranscriptionally regulate gene expression through ؊1 PRF.Programmed ribosomal frameshifting (PRF) is a posttranscriptional regulatory mechanism in which elongating ribosomes are directed to shift the reading frame in response to specific cis-acting signals in mRNAs. Many viruses, including human immunodeficiency virus type 1, use PRF to optimize the stoichiometric ratios between their structural and enzymatic proteins (5, 11). Changes in PRF efficiency alter those ratios, inhibiting virion morphogenesis (9). In particular, retroviruses appear to be very susceptible to such changes (4, 21, 33, 37). Thus, elucidation of the molecular mechanisms underlying PRF can aid in the rational design of antiviral therapeutics (reviewed in reference 8). Though first discovered in viruses, it is also becoming apparent that programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) is also used to regulate expression of cellular genes (reviewed in reference 34). Given the widespread use of PRF, it is important to understand the molecular mechanisms underlying PRF and to identify cellular factors that might be used to regulate these processes.Ribosomes can be directed to shift by one base in either the 5Ј or 3Ј direction depending on the cis-acting signal. Programmed Ϫ1 ribosomal frameshifting is the result of a net shift of the translational reading frame by 1 base in the 5Ј direction. The shift is typically directed by a tripartite cis-acting mRNA element composed of a (from 5Ј to 3Ј) heptameric slippery site, a spacer, and a thermodynamically stable structure, typically an mRNA pseudoknot (reviewed in reference 5). It is generally accepted that Ϫ1 PRF requires a change in the forward kinetics...

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Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-1 gag and pol gene products in stably transfected rodent cell lines

Cited by 14 publications

References 52 publications

A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells

A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells

Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed −1 Ribosomal Frameshifting

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