scholarly journals Viral Translation Is Coupled to Transcription in Sindbis Virus-Infected Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7061-7068 ◽  
Author(s):  
Miguel A. Sanz ◽  
Alfredo Castelló ◽  
Luis Carrasco
Keyword(s):  
Sindbis Virus ◽  
Late Phase ◽  
Mrna Translation ◽  
Encephalomyocarditis Virus ◽  
Host Protein ◽  
Viral Mrnas ◽  
Viral Translation ◽  
Bhk Cells ◽  
Infected Cells

ABSTRACT During the late phase of Sindbis virus infection, the viral subgenomic mRNA is translated efficiently in BHK cells, whereas host protein synthesis is inhibited. However, transfection of in vitro-generated Sindbis virus subgenomic mRNA leads to efficient translation in uninfected BHK cells, whereas it is a poor substrate in infected cells. Therefore, the structure of the subgenomic mRNA itself is not sufficient to confer its translatability in infected cells. In this regard, translation of the subgenomic mRNA requires synthesis from the viral transcription machinery. The lack of translation of transfected viral mRNAs in infected cells is not due to their degradation nor is it a consequence of competition between viral transcripts and transfected mRNAs, because a replicon that cannot produce subgenomic mRNA also interferes with exogenous mRNA translation. Interestingly, subgenomic mRNA is translated more efficiently when it is transfected into uninfected cells than when it is transcribed from a transfected replicon. Finally, a similar behavior was observed for other RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus. These findings support the notion that translation is coupled to transcription in cells infected with different animal viruses.

scholarly journals Preferential Translation of Vesicular Stomatitis Virus mRNAs Is Conferred by Transcription from the Viral Genome

2006 ◽  
Vol 80 (23) ◽  
pp. 11733-11742 ◽  
Author(s):  
Zackary W. Whitlow ◽  
John H. Connor ◽  
Douglas S. Lyles
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Host Protein ◽  
Translation Efficiency ◽  
Viral Mrnas ◽  
Translation Inhibition ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

scholarly journals Analysis of the Adenovirus E1B-55K-Anchored Proteome Reveals Its Link to Ubiquitination Machinery

2002 ◽  
Vol 76 (18) ◽  
pp. 9194-9206 ◽  
Author(s):  
Josephine N. Harada ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
David C. Pallas ◽  
Arnold J. Berk
Keyword(s):  
Ubiquitin Ligase ◽  
Late Phase ◽  
Adenovirus Type ◽  
Nucleocytoplasmic Transport ◽  
Specific Protein ◽  
Viral Mrnas ◽  
Cullin 5 ◽  
Associated Proteins ◽  
Adenovirus E1b

ABSTRACT During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-α1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

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 Leishmania EF-1α Activates the Src Homology 2 Domain Containing Tyrosine Phosphatase SHP-1 Leading to Macrophage Deactivation

2002 ◽  
Vol 277 (51) ◽  
pp. 50190-50197 ◽  
Author(s):  
Devki Nandan ◽  
Taolin Yi ◽  
Martin Lopez ◽  
Crystal Lai ◽  
Neil E. Reiner
Keyword(s):  
Leishmania Donovani ◽  
Tyrosine Phosphatase ◽  
Elongation Factor ◽  
Host Protein ◽  
Src Homology 2 ◽  
Infected Cells ◽  
Src Homology ◽  
Src Homology 2 Domain

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genusLeishmania.The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates ofLeishmania donovanipromastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification ofLeishmaniaelongation factor-1α (EF-1α) as a SHP-1-binding protein. PurifiedLeishmaniaEF-1α, but not host cell EF-1α, bound directly to SHP-1in vitroleading to its activation. Three independent lines of evidence indicated thatLeishmaniaEF-1α may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [35S]methionine-labeled organisms containedLeishmaniaEF-1α. Second, confocal, fluorescence microscopy usingLeishmania-specific antisera detectedLeishmaniaEF-1α in the cytosol of infected cells. Third, co-immunoprecipitation showed thatLeishmaniaEF-1α was associated with SHP-1in vivoin infected cells. Finally, introduction of purifiedLeishmaniaEF-1α, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-γ. Thus,LeishmaniaEF-1α is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.

scholarly journals Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

2000 ◽  
Vol 74 (11) ◽  
pp. 5053-5065 ◽  
Author(s):  
Jeannie F. Spagnolo ◽  
Brenda G. Hogue
Keyword(s):  
Protein Interactions ◽  
Helper Virus ◽  
Host Protein ◽  
Genome Replication ◽  
Mobility Shift ◽  
Bovine Coronavirus ◽  
Replication Assay ◽  
Infected Cells ◽  
Gel Mobility Shift

ABSTRACT RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.

scholarly journals Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Keisuke Nakagawa ◽  
Krishna Narayanan ◽  
Masami Wada ◽  
Shinji Makino
Keyword(s):  
Middle East ◽  
Virus Replication ◽  
Stress Responses ◽  
Middle East Respiratory Syndrome ◽  
Stress Granule ◽  
Accessory Protein ◽  
Viral Mrnas ◽  
Translation Arrest ◽  
Viral Translation ◽  
Infected Cells

ABSTRACTStress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.IMPORTANCEMiddle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

scholarly journals Viral Ribonucleoprotein Complex Formation and Nucleolar-Cytoplasmic Relocalization of Nucleolin in Poliovirus-Infected Cells

1998 ◽  
Vol 72 (8) ◽  
pp. 6699-6709 ◽  
Author(s):  
Shelly Waggoner ◽  
Peter Sarnow
Keyword(s):  
Functional Role ◽  
Infectious Virus ◽  
Viral Gene Expression ◽  
Host Protein ◽  
Viral Gene ◽  
Rna Molecules ◽  
Defective Mutant ◽  
Infected Cells ◽  
Type 3

ABSTRACT The poliovirus 3′ noncoding region (3′NCR) is involved in the efficient synthesis of viral negative-stranded RNA molecules. A strong interaction between a 105-kDa host protein and the wild-type 3′NCR, but not with a replication-defective mutant 3′NCR, was detected. This 105-kDa protein was identified as nucleolin which predominantly resides in the nucleolus and has been proposed to function in the folding of rRNA precursor molecules. A functional role for nucleolin in viral genome amplification was examined in a cell-free extract which has been shown to support the assembly of infectious virus from virion RNA. At early times of viral gene expression, extracts depleted of nucleolin produced less infectious virus than extracts depleted of fibrillarin, another resident of the nucleolus, indicating a functional role of nucleolin in the early stages of the viral life cycle in this in vitro system. Immunofluorescence analysis of uninfected and infected cells showed a nucleocytoplasmic relocalization of nucleolin, but not of fibrillarin, in poliovirus-infected cells. Relocalization of nucleolin was not simply a consequence of virally induced inhibition of translation or transcription, because inhibitors of translation or transcription did not induce nucleolar-cytoplasmic relocalization of nucleolin. These findings suggest a novel virus-induced mechanism by which certain nucleolar proteins are selectively redistributed in infected cells.

scholarly journals A Comparative Quantitative Proteomic Analysis of HCMV-Infected Cells Highlights pUL138 as a Multifunctional Protein

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2520 ◽  
Author(s):  
Yang Li ◽  
Weijuan Shang ◽  
Gengfu Xiao ◽  
Lei-Ke Zhang ◽  
Congyi Zheng
Keyword(s):  
Proteomic Analysis ◽  
Hcmv Infection ◽  
Latent Infection ◽  
Severe Disease ◽  
Late Phase ◽  
Reduction Process ◽  
Host Cells ◽  
Quantitative Proteomic Analysis ◽  
Infected Cells

Human cytomegalovirus (HCMV) is a widespread virus that can establish life-long latent infection in large populations. The establishment of latent infection prevents HCMV from being cleared by host cells, and HCMV reactivation from latency can cause severe disease and death in people with immature or compromised immune systems. To establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by the UL133-UL138 polycistronic locus promotes latent infection in primary CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In this study, recombinant HCMV HanUL138del was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and HanUL138del-infected MRC5 cells was performed to study the effect of pUL138 on host cells in the context of HCMV infection. Our results indicated that, during the early phase of HCMV infection, the innate immune response was differentially activated, while during the late phase of HCMV infection, multiple host proteins were differentially expressed between Han- and HanUL138del-infected cells, and these proteins are involved in the oxidation-reduction process, ER to Golgi vesicle-mediated transport, and extracellular matrix organization. Among these proteins, STEAP3, BORCS7, FAM172A, RELL1, and WDR48 were further demonstrated to affect HCMV infection. Our study provides a systematic view of the effect of pUL138 on the host cell proteome and highlights the proposition that multiple biological processes or host factors may be involved in the overall role of the UL133-UL138 polycistronic locus in HCMV persistence.

scholarly journals The 3′ Untranslated Region of Sindbis Virus Represses Deadenylation of Viral Transcripts in Mosquito and Mammalian Cells

2007 ◽  
Vol 82 (2) ◽  
pp. 880-892 ◽  
Author(s):  
Nicole L. Garneau ◽  
Kevin J. Sokoloski ◽  
Mateusz Opyrchal ◽  
C. Preston Neff ◽  
Carol J. Wilusz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Untranslated Region ◽  
Sindbis Virus ◽  
Mrna Decay ◽  
Mosquito Cell ◽  
Productive Infection ◽  
Temperature Sensitive ◽  
Viral Mrnas ◽  
Cellular Mrnas

ABSTRACT The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5′ cap and a 3′ poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3′ untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3′ UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors—including a 38-kDa protein—were implicated in the repression of deadenylation mediated by the SINV 3′ UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3′ UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.

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