scholarly journals Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

2006 ◽  
Vol 80 (1) ◽  
pp. 360-371 ◽  
Author(s):  
Dorothea L. Sawicki ◽  
Silvia Perri ◽  
John M. Polo ◽  
Stanley G. Sawicki
Keyword(s):  
Host Response ◽  
Rna Synthesis ◽  
Late Phase ◽  
Host Cells ◽  
Minus Strand ◽  
Wild Type ◽  
Persistent Infections ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Transcription Complexes

ABSTRACT In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ∼10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

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.

Persistent Infection of Mouse Fibroblasts (L Cells) with Chlamydia psittaci : Evidence for a Cryptic Chlamydial Form

1980 ◽  
Vol 30 (3) ◽  
pp. 874-883
Author(s):  
James W. Moulder ◽  
Nancy J. Levy ◽  
Laura P. Schulman
Keyword(s):  
Host Cell ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Developmental Cycle ◽  
Persistently Infected ◽  
Mouse Fibroblasts ◽  
Persistent Infections ◽  
L Cells ◽  
Infected Cells ◽  
Parasite Multiplication

When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 10 5 to 10 6 originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis . These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.

scholarly journals Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication

2004 ◽  
Vol 78 (11) ◽  
pp. 5957-5965 ◽  
Author(s):  
Mark R. Denison ◽  
Boyd Yount ◽  
Sarah M. Brockway ◽  
Rachel L. Graham ◽  
Amy C. Sims ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Wild Type ◽  
Virus Growth ◽  
Amino Terminal ◽  
Normal Expression ◽  
Infected Cells ◽  
Critical Residues

ABSTRACT The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), 247Gly↓Val248, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

scholarly journals Role of Tachykinins in the Host Response to Murine Gammaherpesvirus Infection

2001 ◽  
Vol 75 (21) ◽  
pp. 10467-10471 ◽  
Author(s):  
Catherine M. Payne ◽  
Caroline J. Heggie ◽  
David G. Brownstein ◽  
James P. Stewart ◽  
John P. Quinn
Keyword(s):  
Virus Infection ◽  
Host Response ◽  
Infectious Virus ◽  
Inflammatory Cells ◽  
Direct Influence ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Tachykinins function not only as neurotransmitters but also as immunological mediators. We used infection of tachykinin-deficient (PPT-A −/−) mice and wild-type controls with murine gammaherpesvirus to assess the role of tachykinins in the host response to a virus infection. Although infection was ultimately controlled in PPT-A −/− mice, there were higher titers of infectious virus in the lungs, accompanied by a more rapid influx of inflammatory cells. Clearance of latently infected cells from the spleen was also delayed. This is the first report of the direct influence of tachykinins in the host response to a virus infection.

scholarly journals An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors

2014 ◽  
Vol 95 (12) ◽  
pp. 2683-2692 ◽  
Author(s):  
Irina C. Albulescu ◽  
Ali Tas ◽  
Florine E. M. Scholte ◽  
Eric J. Snijder ◽  
Martijn J. van Hemert
Keyword(s):  
Mode Of Action ◽  
Chikungunya Virus ◽  
Rna Synthesis ◽  
Mechanistic Studies ◽  
Vitro Assay ◽  
Virus Rna ◽  
Infected Cells ◽  
Transcription Complexes ◽  
Crude Membrane Fraction

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

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 Loss of Virulence of the Phytopathogen Ralstonia solanacearum Through Infection by φRSM Filamentous Phages

2012 ◽  
Vol 102 (5) ◽  
pp. 469-477 ◽  
Author(s):  
Hardian S. Addy ◽  
Ahmed Askora ◽  
Takeru Kawasaki ◽  
Makoto Fujie ◽  
Takashi Yamada
Keyword(s):  
Ralstonia Solanacearum ◽  
Virulence Genes ◽  
Host Cells ◽  
Extracellular Polysaccharides ◽  
Twitching Motility ◽  
Wild Type ◽  
Tomato Plants ◽  
Type Iv ◽  
Infected Cells ◽  
Filamentous Phages

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

scholarly journals Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase

2002 ◽  
Vol 76 (17) ◽  
pp. 8632-8640 ◽  
Author(s):  
Cori L. Fata ◽  
Stanley G. Sawicki ◽  
Dorothea L. Sawicki
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Temperature Sensitive ◽  
Minus Strand ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Lethal Temperature ◽  
Host Restriction ◽  
Mosquito Cells ◽  
Promoter Recognition

ABSTRACT A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.

scholarly journals The Stability of the Duplex between Sense and Antisense Transcription-Regulating Sequences Is a Crucial Factor in Arterivirus Subgenomic mRNA Synthesis

2003 ◽  
Vol 77 (2) ◽  
pp. 1175-1183 ◽  
Author(s):  
Alexander O. Pasternak ◽  
Erwin van den Born ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Proximal Part ◽  
The Body ◽  
Equine Arteritis Virus ◽  
Strand Transfer ◽  
Minus Strand ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Stability

ABSTRACT Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a “body” part of variable size, which are derived from the 5′- and 3′-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus subgenomic mRNA synthesis.

scholarly journals Baculovirus lef-12 Is Not Required for Viral Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12032-12043 ◽  
Author(s):  
Linda A. Guarino ◽  
Toni-Ann Mistretta ◽  
Wen Dong
Keyword(s):  
Trichoplusia Ni ◽  
Late Phase ◽  
Growth Curves ◽  
Single Step ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Step Growth

ABSTRACT The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.

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