Use of Eukaryotic Native Small Ribosomal Subunits for the Translation of Globin Messenger RNA

Freienstein, Christoph; Blobel, Günter

doi:10.1073/pnas.71.9.3435

Cited by 24 publications

(11 citation statements)

References 18 publications

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“…The first evidence that immature HIV-1 capsids assemble via a host-catalyzed pathway of intermediates was generated in a cell-free system. Adapted from the in vitro translation systems that were used to identify signal sequences (32), this cell-free assembly system supports de novo synthesis of HIV-1 Gag from a Gag mRNA using energy substrates, amino acids, and a cellular extract that provides host factors required for Gag translation as well as post-translational events of Gag assembly. When programmed with wild-type Gag mRNA, this system produces particles that closely resemble completed immature HIV-1 capsids produced by HIV-1 produced by provirus-expressing cells in their ultrastructural details as well as in their size and shape (as defined by a sedimentation value of ~750S; (24)).…”

Section: Resultsmentioning

confidence: 99%

Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

Reed

Solas²,

Kitaygorodskyy³

et al. 2020

Preprint

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Given the projected increase in multidrug resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life-cycle stages that are not targeted by antiretrovirals currently in use. Host-targeting drugs are of particular interest because they can offer a high barrier to resistance. Here we report identification of two related small molecules that inhibit HIV-1 late events, a stage of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and PBMCs and are effective against a primary isolate. They reduce virus production, likely by inhibiting a post-translational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed in a parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds might localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that are formed during immature HIV-1 capsid assembly. Indeed, imaging of infected cells showed colocalization of the compound with two host enzymes found in assembly intermediates, ABCE1 and DDX6. While the exact target and mechanism of action of this chemotype remain to be determined, these findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.IMPORTANCEThe success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including viral-host interactions that promote assembly. This effort led to identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations by inhibiting virus production. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly but resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype may represent a first-in-class inhibitor of virus production that acts by targeting a viral-host complex important for HIV-1 Gag assembly.

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Section: Resultsmentioning

confidence: 99%

Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

Reed

Solas²,

Kitaygorodskyy³

et al. 2020

Preprint

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“…Preparations of native small ribosomal subunits (S t~) from rabbit reticulocytes, of derived large ribosomal subunits (L ~ from rat liver free ribosomes by the puromycin-KCI procedure, and of pH 5 enzymes from a high speed supernate of Krebs ascites cells were as previously described (6,13).…”

Section: Fractionation Of Mopc 41 Dl-i Tumormentioning

confidence: 99%

Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

Blobel¹,

Dobberstein²

1975

The Journal of Cell Biology

2,927

1,151

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Fractionation of MOPC 41 DL-I tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a beterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same tool wt as the precursor of the light chain.In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain.The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains.

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“…Samples were prepared as described (3). Gradient Sn fraction into a sucrose gradient containing 500 mM KC1, buffer, and 5 mM MgCl2 resulted in a dominant peak sedimenting at 40S (labeled S in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide.…”

Section: Methodsmentioning

confidence: 99%

“…In eukaryotes it was recently established that the so-called native small ribosomal subunits (Sn), which comprise only a few percent of the total ribosomes, are able to initiate translation of mRNA in a reconstituted system containing derived large ribosomal subunits and pH 5 enzymes as the only other components (3,4). Furthermore, it was reported that Sn contained several nonribosomal proteins (5) and that a salt extract from sn could stimulate globin mRNA translation in a cell-free protein synthesis system (6).…”

mentioning

confidence: 99%

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Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

Freienstein¹,

Blobel²

1975

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

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The native small ribosomal subunit (S9) from rabbit reticulocytes which is able to initiate translation of globin mRNA in a cell-free system carries additional protein components. The latter can be separated from the subunit in a high salt sucrose gradient yielding a top-fraction (7) and a complex fraction (C), sedimenting at about 4 and 15 S, respectively. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that fraction T contained four dominant polypeptides, while fraction C represents a large protein complex consisting of at least 10 polypeptides. SI' isolated from other sources showed similar patterns of their nonribosomal proteins.Reconstitution experiments revealed that fraction C is absolutely required for protein synthesis, while fraction T enhances protein synthesis only in the presence of C.The adherence of these protein factors to the subunit is not mediated by magnesium ions. Treatment of SO with EDTA and centrifugation in a magnesium-free sucrose gradient caused unfolding of the subunits and dissociation of several ribosomal proteins, but not of the factors. The unfolded ribosomal subunits sedimented as two distinct peaks. The more slowly sedimenting peak contained proteins of fraction T and the faster sedimenting one contained the 15S complex, indicating heterogeneity of the 9' population with respect to the factors attached to them.Initiation factors for protein synthesis in pro-and eukaryotes are in general found to be bound to ribosomes, from which they can be extracted by high concentrations of monovalent ions (1, 2). In eukaryotes it was recently established that the so-called native small ribosomal subunits (Sn), which comprise only a few percent of the total ribosomes, are able to initiate translation of mRNA in a reconstituted system containing derived large ribosomal subunits and pH 5 enzymes as the only other components (3, 4). Furthermore, it was reported that Sn contained several nonribosomal proteins (5) and that a salt extract from sn could stimulate globin mRNA translation in a cell-free protein synthesis system (6).These findings prompted us to identify the nonribosomal proteins found in Sn and to investigate their possible localization in other components of the ribosomal fraction, such as monosomes and polysomes, their mode of linkage to the small ribosomal subunit, their occurrence in a variety of cells, and their role in a reconstituted system. METHODSPreparation of Native Small Ribosomal Subunits (9'). Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide. Further operations were performed in the cold (1-4°). Washed reticulocytes were lysed in 2 volumes of a solution of 10 mM Tris-HCl (pH 7.5)-10 mM KC1-1.5 mM MgCl2-2 mM dithiothreitol and homogenized with three strokes in a Potter-Elvehjem homogenizer. The postmitochondrial supernatant (10 min, 20,000 X gav) was centrifuged first for 30 min and the resulting supernatant was subsequently centrifuged again for 5 hr at 105,0...

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Use of Eukaryotic Native Small Ribosomal Subunits for the Translation of Globin Messenger RNA

Cited by 24 publications

References 18 publications

Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

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