Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

Boone, Lawrence R.; Skalka, A M

doi:10.1073/pnas.77.2.847

Cited by 33 publications

(24 citation statements)

References 41 publications

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“…The structural orientation of M in the intact virion is such that this protein interacts with both the glycoprotein G and the RNP protein N; evidence for this statement is based on the initial observations of VSV protein cross-linking with formalin (Brown et al, 1974) and later studies that demonstrated the formation of G :M and M :N dimers and in the presence of cross-linking agents (Dubovi & Wagner, 1977;Mudd & Swanson, 1978). Phosphorylation of M may provide a mechanism for the disruption of these interactions during uncoating.…”

Section: Discussionmentioning

confidence: 99%

“…When used in an in vitro transcription reaction mixture, melittin, the 26 amino acid polypeptide active component of bee venom, has been reported to activate the synthesis of VSV mRNA and leader RNA (Boone & Skalka, 1980). Melittin interacts with biological membranes to produce localized perturbations in the bilayer structure at sites of insertion (Williams & Bell, 1972) and, therefore, does not solubilize membranes as do non-ionic detergents.…”

Section: Activation Of the In Vitro Vs V Kinase And Transcription Reamentioning

confidence: 99%

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Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

Witt¹,

Naeve²,

Summers³

1981

Journal of General Virology

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SUMMARYThe relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of y-[32p]ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observati~n. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

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Section: Discussionmentioning

confidence: 99%

Section: Activation Of the In Vitro Vs V Kinase And Transcription Reamentioning

confidence: 99%

Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

Witt¹,

Naeve²,

Summers³

1981

Journal of General Virology

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“…ERT assays. Endogenous RT (ERT) reactions were performed as previously described (7,8), with modifications as described below. Equivalent amounts of virus particles (normalized by RT content) were incubated with melittin (100 g/ml, final concentration) at 41°C for 10 min prior to addition of ERT buffer (0.1 M Tris-HCl [pH 8.3], 25 mM NaCl, 3 mM magnesium acetate, 30 mM dithiothreitol, 1 mM dCTP, 1 mM dATP, 1 mM dGTP [final concentrations]).…”

Section: Methodsmentioning

confidence: 99%

RNA Dimerization Defect in a Rous Sarcoma Virus Matrix Mutant

Parent

Cairns

Albert³

et al. 2000

J Virol

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The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown to contain functions required for membrane targeting and binding during particle assembly and budding. Additional functions for MA have been proposed based on the existence of MA mutants in Rous sarcoma virus (RSV), murine leukemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of normal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small membrane-binding domain of the Src oncoprotein has been added as an N-terminal extension of Gag. While Myr1E is not infectious, full infectivity can be reestablished by a single amino acid substitution in the Src sequence (G2E), which eliminates the addition of myristic acid and the membrane-binding capacity of this foreign sequence. The presence of myristic acid at the N terminus of the Myr1E Gag protein does not explain its replication defect, because other myristylated derivatives of RSV Gag are fully infectious (e.g., Myr2 [C. R. Erdie and J. W. Wills, J. Virol. 64:5204-5208, 1990]). Biochemical analyses of Myr1E particles reveal that they contain wild-type levels of the Gag cleavage products, Env glycoproteins, and reverse transcriptase activity when measured on an exogenous template. Genomic RNA incorporation appears to be mildly reduced compared to the wild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturing Northern blots. Importantly, the insertional mutation does not lie within previously identified dimer linkage sites. In spite of the dimerization defect, the genomic RNA from Myr1E particles serves efficiently as a template for reverse transcription as measured by an endogenous reverse transcriptase assay. In marked contrast, after infection of avian cells, the products of reverse transcription are nearly undetectable. These findings might be explained either by the loss of a normal function of MA needed in the formation or stabilization of RNA dimers or by the interference in such events by the mutant MA molecules. It is possible that Myr1E viruses package a single copy of viral RNA.The retrovirus Gag polyprotein directs the assembly and budding of virus particles from the plasma membrane of infected cells. Extensive functional mapping of several Gag proteins has led to the identification of three common assembly domains that are required for this activity. The N-terminal membrane-binding (M) domain directs Gag molecules from the cytoplasm to the inner leaflet of the plasma membrane (44,55,63) where aggregates of Gag proteins are formed through protein-protein interactions primarily involving the I (interaction) domains (4, 12, 22, 57). These interactions lead to the emergence of spherical particles that pinch off the membrane during the final step in the budding process, which is mediated by the L (late) assembly domain (27,45,58). Virus maturation occurs as the...

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“…We also tested melittin, an amphipathic polypeptide derived from bee venom which forms membrane pores with an average size of 3 nm and can accommodate the release of 50.7-kDa size markers from permeabilized vesicles (34). Melittin has been reported to allow synthesis of more full-length cDNA in the endogenous RT reaction compared to nonionic detergents (4,6,14,66).…”

Section: Detergent Stability Of Virionsmentioning

confidence: 99%

Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core

Öhagen

Gabuzda

2000

J Virol

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The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription. vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.

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Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

Cited by 33 publications

References 41 publications

Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

RNA Dimerization Defect in a Rous Sarcoma Virus Matrix Mutant

Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core

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