scholarly journals Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

1985 ◽  
Vol 5 (11) ◽  
pp. 2913-2923 ◽  
Author(s):  
H D Bernstein ◽  
N Sonenberg ◽  
D Baltimore
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Hela Cells ◽  
Mrna Translation ◽  
Selective Inhibition ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Amino Terminal ◽  
Translational Inhibition

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.

scholarly journals Poliovirus mutant that does not selectively inhibit host cell protein synthesis

1985 ◽  
Vol 5 (11) ◽  
pp. 2913-2923 ◽  
Author(s):  
H D Bernstein ◽  
N Sonenberg ◽  
D Baltimore
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Hela Cells ◽  
Mrna Translation ◽  
Selective Inhibition ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Amino Terminal ◽  
Translational Inhibition

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.

scholarly journals Infection of HeLa cells with Chlamydia trachomatis inhibits protein synthesis and causes multiple changes to host cell pathways

2019 ◽  
Vol 21 (4) ◽  
Author(s):  
Michaela Ohmer ◽  
Tina Tzivelekidis ◽  
Nora Niedenführ ◽  
Larisa Volceanov‐Hahn ◽  
Svenja Barth ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Hela Cells

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

scholarly journals Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein

2012 ◽  
Vol 93 (7) ◽  
pp. 1483-1494 ◽  
Author(s):  
Michelle M. Arnold ◽  
Catie Small Brownback ◽  
Zenobia F. Taraporewala ◽  
John T. Patton
Keyword(s):  
Binding Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Nuclear Accumulation ◽  
Progeny Virus ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Infected Cells

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3′-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines.

scholarly journals UL54-Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture

2006 ◽  
Vol 80 (2) ◽  
pp. 769-784 ◽  
Author(s):  
Jennifer A. Schwartz ◽  
Elizabeth E. Brittle ◽  
Ashley E. Reynolds ◽  
Lynn W. Enquist ◽  
Saul J. Silverstein
Keyword(s):  
Protein Synthesis ◽  
Pseudorabies Virus ◽  
Host Protein ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type ◽  
In Cells

ABSTRACT The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and λRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJSΔ54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSΔ54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSΔ54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJSΔ54. Interestingly, vJSΔ54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSΔ54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.

Transcriptional and translational events during reovirus infection

1988 ◽  
Vol 66 (8) ◽  
pp. 803-812 ◽  
Author(s):  
Guy Lemay
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Mrna Translation ◽  
Cell Membrane Permeability ◽  
Cell Protein ◽  
Viral Mrna ◽  
Genomic Rna ◽  
Host Cell Protein ◽  
Outer Capsid ◽  
Viral Genomic Rna

This short review focuses on the mechanisms involved in transcription and translation in mouse L cells infected with reoviruses. The viral genomic RNA (double-stranded), retained in the inner capsid following removal of the outer capsid of the infecting virion, is transcribed by a viral polymerase. The synthesized viral mRNA is blocked at the 5′ end by a cap structure similar to the cap structure of cellular mRNA but synthesized by the viral enzymes of the inner capsid. This viral mRNA is also used as the first strand and template for the synthesis of the second strand of viral genomic RNA; the newly replicated genome is retained in an inner capsid structure to generate the progeny subviral particles. These particles are active at the transcriptional level but do not synthesize the cap, owing to the absence of the guanylyltransferase activity involved in the formation of this structure. The uncapped mRNA, or late viral mRNA, constitutes the bulk part of viral mRNA. The transcription of the viral genome is finally arrested upon addition of outer capsid proteins to obtain a mature virion. During viral multiplication, there is a gradual inhibition of host-cell protein synthesis, concomitant with stimulation of late viral mRNA translation. The two phenomena are apparently distinct. Furthermore, the inhibition of host-cell protein synthesis has been shown to be dispensable for normal virus multiplication; however, it might accelerate it. The mechanisms responsible for inhibition are still unclear but might involve modifications in the activity of cellular cap-binding proteins. This last point suggests an analogy with poliovirus infection; the two systems are thus briefly compared. Possible significance of the absence of a poly(A) tract at the 3′ end of reovirus mRNA, in contrast to the occurrence of such a sequence at the end of cellular mRNA, is also examined. Different models involving cap discrimination, competition between mRNAs, or alteration of cell membrane permeability have been proposed to explain the events observed at the translational level in reovirus-infected cells. These different models are compared. Finally, recent data implicating the viral sigma 3 capsid protein in efficient translation of late viral mRNA are discussed.

scholarly journals The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.

1986 ◽  
Vol 6 (2) ◽  
pp. 470-476 ◽  
Author(s):  
S Pilder ◽  
M Moore ◽  
J Logan ◽  
T Shenk
Keyword(s):  
Hela Cells ◽  
Adenovirus Type ◽  
Transcription Unit ◽  
Early Gene ◽  
Wild Type Virus ◽  
Wild Type ◽  
Coding Region ◽  
Viral Mrnas ◽  
Cellular Mrnas ◽  
Adenovirus Type 5

The adenovirus type 5 mutant H5dl338 lacks 524 base pairs within early region 1B. The mutation removed a portion of the region encoding the related E1B-55K and -17K polypeptides but did not disturb the E1B-21K coding region. The virus can be propagated in 293 cells which contain and express the adenovirus type 5 E1A and E1B regions, but it is defective for growth in HeLa cells, in which its final yield is reduced about 100-fold compared with the wild-type virus. The mutant also fails to transform rat cells at normal efficiency. The site of the dl338 defect was studied in HeLa cells. Early gene expression and DNA replication appeared normal. Late after infection, mRNAs coded by the major late transcription unit accumulated to reduced levels. At a time when transcription rates and steady-state nuclear RNA species were normal, the rate at which late mRNA accumulated in the cytoplasm was markedly reduced. Furthermore, in contrast to the case with the wild type, transport and accumulation of cellular mRNAs continued late after infection with dl338. Thus, the E1B product appears to facilitate transport and accumulation of viral mRNAs late after infection while blocking the same processes for cellular mRNAs.

scholarly journals Polyomavirus Small T Antigen Controls Viral Chromatin Modifications through Effects on Kinetics of Virus Growth and Cell Cycle Progression

2007 ◽  
Vol 81 (18) ◽  
pp. 10064-10071 ◽  
Author(s):  
Jean Dahl ◽  
H. Isaac Chen ◽  
Michael George ◽  
Thomas L. Benjamin
Keyword(s):  
Cell Cycle ◽  
Histone Modifications ◽  
Cell Cycle Progression ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Type Virus ◽  
Wild Type ◽  
T Antigens ◽  
Cycle Progression ◽  
Virus Growth

ABSTRACT Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G2 and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type- and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominately in G2. These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.

scholarly journals Cleavage Specificity of the UL48 Deubiquitinating Protease Activity of Human Cytomegalovirus and the Growth of an Active-Site Mutant Virus in Cultured Cells

2009 ◽  
Vol 83 (23) ◽  
pp. 12046-12056 ◽  
Author(s):  
Eui Tae Kim ◽  
Se Eun Oh ◽  
Yun-Ok Lee ◽  
Wade Gibson ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Active Site ◽  
Protease Activity ◽  
Cultured Cells ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Type Virus ◽  
Wild Type ◽  
Cleavage Specificity

ABSTRACT The human cytomegalovirus (HCMV) open reading frame UL48 encodes a 253-kDa tegument protein that is closely associated with the capsid and was recently shown to have ubiquitin-specific protease activity (J. Wang, A. N. Loveland, L. M. Kattenhorn, H. L. Ploegh, and W. Gibson, J. Virol. 80:6003-6012, 2006). Here, we examined the cleavage specificity of this deubiquitinase (DUB) and replication characteristics of an active-site mutant virus. The purified catalytic domain of the UL48 DUB (1 to 359 amino acids), corresponding to the herpes simplex virus UL36USP DUB (L. M. Kattenhorn, G. A. Korbel, B. M. Kessler, E. Spooner, and H. L. Ploegh, Mol. Cell 19:547-557, 2005), efficiently released ubiquitin but not ubiquitin-like modifications from a hemagglutinin peptide substrate. Mutating the active-site residues Cys24 or His162 (C24S and H162A, respectively) abolished this activity. The HCMV UL48 and HSV UL36USP DUBs cleaved both Lys48- and Lys63-linked ubiquitin dimers and oligomers, showing more activity toward Lys63 linkages. The DUB activity of the full-length UL48 protein immunoprecipitated from virus-infected cells also showed a better cleavage of Lys63-linked ubiquitinated substrates. An HCMV (Towne) mutant virus in which the UL48 DUB activity was destroyed [UL48(C24S)] produced 10-fold less progeny virus and reduced amounts of viral proteins compared to wild-type virus at a low multiplicity of infection. The mutant virus also produced perceptibly less overall deubiquitination than the wild-type virus. Our findings demonstrate that the HCMV UL48 DUB contains both a ubiquitin-specific carboxy-terminal hydrolase activity and an isopeptidase activity that favors ubiquitin Lys63 linkages and that these activities can influence virus replication in cultured cells.

scholarly journals EXPRESSION OF THE MITOCHONDRIAL GENOME IN HELA CELLS

1973 ◽  
Vol 56 (3) ◽  
pp. 819-831 ◽  
Author(s):  
Brian Storrie ◽  
Giuseppe Attardi
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Genome ◽  
Hela Cells ◽  
Mitochondrial Protein ◽  
Selective Inhibition ◽  
Mitochondrial Protein Synthesis ◽  
Cloning Efficiency ◽  
Cell Growth Inhibition ◽  
Progressive Loss ◽  
Two Generations

The effect of selective inhibition of mitochondrial protein synthesis by chloramphenicol at 40 or 200 µg/ml on the formation of mitochondria in HeLa cells was investigated. HeLa cells, under the conditions used in the present work, grow at a decreasing rate for at least four cell generations in the presence of 40 µg/ml chloramphenicol, and for two generations in the presence of 200 µg/ml chloramphenicol. The progressive cell growth inhibition which begins after 2 days of exposure of the cells to 40 µg/ml chloramphenicol is immediately or gradually reversible, upon removal of the drug, for periods up to at least 8 days of treatment, though there is a progressive loss of cloning efficiency. In cells which have been treated for 6–7 days with 40 or 200 µg/ml of chloramphenicol, mitochondrial protein synthesis occurs at a normal or near-normal rate 1 h after removal of the drug. Mitochondria increase normally in number and show a normal size and amount of cristae in the presence of either concentration of drug. However, in 4–5% of the mitochondrial profiles the cristae appear to be arranged in unusual, circular, looped or whorled configuration.

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