scholarly journals Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

2001 ◽  
Vol 75 (24) ◽  
pp. 12153-12160 ◽  
Author(s):  
Chih-Wen Peng ◽  
Valera V. Peremyslov ◽  
Arcady R. Mushegian ◽  
William O. Dawson ◽  
Valerian V. Dolja
Keyword(s):  
Cell Movement ◽  
Duplication Event ◽  
Functional Specialization ◽  
Virus Rna ◽  
The Family ◽  
Lettuce Infectious Yellows Virus ◽  
Rna Accumulation ◽  
Positive Strand Rna ◽  
L1 And L2

ABSTRACT Members of the Closteroviridae andPotyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus(a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

scholarly journals Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
Author(s):  
Marco Binder ◽  
Doris Quinkert ◽  
Olga Bochkarova ◽  
Rahel Klein ◽  
Nikolina Kezmic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Negative Strand ◽  
Virus Rna ◽  
Nontranslated Region ◽  
Genotype 2 ◽  
Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

scholarly journals Size-dependent cell-to-cell movement of defective interfering RNAs of Cymbidium ringspot virus

2002 ◽  
Vol 83 (6) ◽  
pp. 1505-1510 ◽  
Author(s):  
György Szittya ◽  
Dániel Silhavy ◽  
Tamás Dalmay ◽  
József Burgyán
Keyword(s):  
Transgenic Plants ◽  
Nicotiana Benthamiana ◽  
Time Course ◽  
De Novo ◽  
Cell Movement ◽  
Ringspot Virus ◽  
Size Dependent ◽  
Dependent Cell ◽  
Rna Accumulation

Co-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.

scholarly journals Interaction of Pregnancy-Specific Glycoprotein 1 With Integrin Α5β1 Is a Modulator of Extravillous Trophoblast Functions

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1369 ◽  
Author(s):  
Rattila ◽  
Dunk ◽  
Im ◽  
Grichenko ◽  
Zhou ◽  
...  
Keyword(s):  
African American Women ◽  
Late Onset ◽  
Cell Movement ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
In Vitro Assays ◽  
The Family ◽  
Oxygen Conditions ◽  
Movement Tracking

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin 5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to 51 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to 51 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy.

scholarly journals The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 1045-fold amplification

2000 ◽  
Vol 81 (9) ◽  
pp. 2307-2311 ◽  
Author(s):  
Masayuki Ishikawa ◽  
Michael Janda ◽  
Paul Ahlquist
Keyword(s):  
Cell Division ◽  
Mosaic Virus ◽  
Rna Virus ◽  
Cell Movement ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Rna ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Rna Replicon

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85–95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura+ colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

scholarly journals Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

2010 ◽  
Vol 84 (9) ◽  
pp. 4229-4242 ◽  
Author(s):  
Kenneth J. Ertel ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Synthesis ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Positive Strand ◽  
Negative Strand ◽  
Virus Rna ◽  
Hnrnp C ◽  
C Proteins ◽  
Positive Strand Rna

ABSTRACT The poliovirus 3′ noncoding region (3′ NCR) is necessary for efficient virus replication. A poliovirus mutant, PVΔ3′NCR, with a deletion of the entire 3′ NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5′ end of poliovirus negative-strand RNA (the complement of the genomic 3′ NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3′ end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5′ end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.

scholarly journals Role of the Alfalfa Mosaic Virus Methyltransferase-Like Domain in Negative-Strand RNA Synthesis

2002 ◽  
Vol 76 (22) ◽  
pp. 11321-11328 ◽  
Author(s):  
A. Corina Vlot ◽  
Aymeric Menard ◽  
John F. Bol
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Alfalfa Mosaic Virus ◽  
Positive Strand ◽  
Negative Strand ◽  
Putative Role ◽  
Associated Functions ◽  
Rna Accumulation ◽  
Positive Strand Rna

ABSTRACT RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

Analysis of the subgenomic RNAs and the small open reading frames of Beet black scorch virus

2006 ◽  
Vol 87 (10) ◽  
pp. 3077-3086 ◽  
Author(s):  
Xuefeng Yuan ◽  
Yunhe Cao ◽  
Dehui Xi ◽  
Lihua Guo ◽  
Chenggui Han ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Essential Gene ◽  
Cell Movement ◽  
Site Directed Mutagenesis ◽  
Chenopodium Amaranticolor ◽  
Viral Rnas ◽  
Local Lesions ◽  
Rna Accumulation ◽  
Beet Black Scorch Virus

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209 within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526 within the nested p7b/p5′ ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5′) encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves of Chenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5′ proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions on C. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.

scholarly journals Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA

2006 ◽  
Vol 87 (9) ◽  
pp. 2605-2614 ◽  
Author(s):  
Indira Umareddy ◽  
Alex Chao ◽  
Aruna Sampath ◽  
Feng Gu ◽  
Subhash G. Vasudevan
Keyword(s):  
Dengue Virus ◽  
Virus Replication ◽  
Physical Interaction ◽  
Helicase Domain ◽  
Virus Infected Cell ◽  
Yeast Two Hybrid ◽  
The Family ◽  
Positive Strand Rna ◽  
Two Hybrid

Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3.

scholarly journals Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713

2006 ◽  
Vol 87 (12) ◽  
pp. 3703-3714 ◽  
Author(s):  
Liying Sun ◽  
Donald L. Nuss ◽  
Nobuhiro Suzuki
Keyword(s):  
Vertical Transmission ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight Fungus ◽  
Virus Rna ◽  
The Family ◽  
Heterologous Virus ◽  
In Trans ◽  
Rna Accumulation ◽  
Suppressor Of Rna Silencing

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys→Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Δp29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys162. These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

scholarly journals Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae

2009 ◽  
Vol 84 (5) ◽  
pp. 2511-2521 ◽  
Author(s):  
Barry R. O'Keefe ◽  
Barbara Giomarelli ◽  
Dale L. Barnard ◽  
Shilpa R. Shenoy ◽  
Paul K. S. Chan ◽  
...  
Keyword(s):  
Immunological Response ◽  
Large Family ◽  
Infection Model ◽  
Human Pathogens ◽  
Emerging Viruses ◽  
Antiviral Protein ◽  
The Family ◽  
Positive Strand Rna

ABSTRACT Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

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