Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Zeng, Chi; Wentz, Melissa J.; Cohen, Jean; Estes, Mary K.; Ramig, Robert F.

doi:10.1128/jvi.70.5.2736-2742.1996

Cited by 83 publications

(51 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Infected cells and cell culture supernatants were harvested separately. Cells were lysed by three cycles of freezing-thawing or by treatment with lysis buffer containing protease inhibitors (58). The treated cell lysates and supernatants were concentrated by ultracentrifugation at 112,700 ϫ g (SW28Ti rotor; Beckman Coulter, Inc., Fullerton, Calif.) for 2 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Self-Assembly of the Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses

et al. 2005

View full text Add to dashboard Cite

None of the enteric caliciviruses except Po/Sapo/GIII/Cowden/80/US replicates in cell culture, which complicates efforts to develop control strategies or to study viral replication. To develop serological assays for bovine noroviruses (BoNVs) and to determine the cross-reactivity of BoNV with human noroviruses, we generated two recombinant baculoviruses, rCV186-OH and rJNCV, to express the capsid genes of Bo/CV186-OH/00/US (

show abstract

Section: Methodsmentioning

confidence: 99%

Self-Assembly of the Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses

et al. 2005

View full text Add to dashboard Cite

show abstract

“…First, the utilization of high MOI, even when large virus stocks are required, guarantees that each cell is infected by at least one pfu of each virus type. This is important, as it has been determined that VP6 is required for VP2 release from membranes (Zeng et al, 1996). Thus, for the assembly of double-shelled RLPs it is necessary that each cell synthesizes both VP2 and VP6.…”

Section: Utility Of Moi To Manipulate the Stoichiometric Relation Betmentioning

confidence: 99%

“…Similar to the case of MOI, the ability of manipulating the VP6/VP2 ratio through the use of nonsimultaneous infections can be a powerful tool for attaining protein concentration ratios that maximize VLP assembly. As mentioned before, synthesis of both VP2 and VP6 within the same cell is necessary for assembly of double-shelled RLP because coexpression is necessary for VP2 solubilization (Zeng et al, 1996). Accordingly, manipulation of TOI will only be useful if the same cells that were initially infected with bac2 are susceptible to a later infection with bac6.…”

Section: The Utility Of Toi To Manipulate the Stoichiometric Relationmentioning

confidence: 99%

Strategies for manipulating the relative concentration of recombinant rotavirus structural proteins during simultaneous production by insect cells

Palomares

López

Ramı́rez

2002

Biotech & Bioengineering

View full text Add to dashboard Cite

Adequate production strategies of virus-like particles are among the challenges that must be addressed before such complex multimeric structures find practical applications as vaccines. Attainment of the correct stoichiometric relation between proteins that constitute virus-like particles should result in an increased productivity by maximizing the concentration of assembled proteins and preventing the accumulation of waste monomers. In this work, strategies for manipulating the relative concentration between two of the structural proteins that constitute rotavirus-like particles (VP2 and VP6) were explored using the insect cell baculovirus expression vector system. It was shown that multiplicity of infection is a useful tool for manipulating protein production rates and maximum concentrations in cultures expressing one or two recombinant proteins. Thus, multiplicity of infection can be employed for improving production of rotavirus-like particles. VP2 and VP6 production rates obtained during individual infections remained unchanged when both were simultaneously produced, indicating that such rates can be utilized for estimating protein concentrations during coexpression. Manipulation of the time of infection between the two recombinant baculoviruses, proposed here for the first time, also proved to be effective for controlling the relative protein concentrations. The use of such sequential infections constituted an effective production alternative that does not require high amounts of virus stocks and is easy to implement. In addition to VP2 and VP6, kinetic parameters for the individual production of the other two proteins (VP4 and VP7) that constitute rotavirus-like particles were also obtained.

show abstract

“…This relationship between transcription and replication is being understood in greater detail from studies conducted using cystovirus ~6 Van Dijk et al, 1995;Qiao et al, 1997;Mindich, 1999), where a complete viral reconstitution system is available to allow recovery of infectious virions from mRNA transcripts or cDNA clones of the viral genome (Olkkonen et al, 1990). Although no reconstitution system is yet available for any of the reoviruses, studies conducted using baculovirusexpressed recombinant rotavirus-like particles containing the viral RNA polymerase coexpressed with the inner capsid protein have begun to clarify the mechanism by which the dsRNA genome is synthesized from the mRNA templates in rotavirus Patton et al, 1996;Zeng et al, 1996).…”

Section: Mature Mrna Transcriptsmentioning

confidence: 99%

Mechanism of genome transcription in segmented dsRNA viruses

Lawton

Estes

Prasad

2000

Advances in Virus Research

View full text Add to dashboard Cite

Genome transcription is a critical stage in the life cycle of a virus, as this is the process by which the viral genetic information is presented to the host cell protein synthesis machinery for the production of the viral proteins needed for genome replication and progeny virion assembly. Viruses with dsRNA genomes face a particular challenge in that host cells do not produce proteins which can transcribe from a dsRNA template. Therefore, dsRNA viruses contain all of the necessary enzymatic machinery to synthesize complete mRNA transcripts within the core without the need for disassembly. Indeed one of the more striking observations about genome transcription in dsRNA viruses is that this process occurs efficiently only when the transcriptionally competent particle is fully intact. This observation suggests that all of the components of the TCP, including the viral genome, the transcription enzymes, and the viral capsid, function together to produce and release mRNA transcripts and that each component has a specific and critical role to play in promoting the efficiency of this process. This review has examined the process of genome transcription in dsRNA viruses from the perspective of rotavirus as a model system. However, despite numerous architectural and organizational differences among the families of dsRNA viruses, numerous studies suggest that the basic mechanism of mRNA production may be similar in most, if not all, viruses having dsRNA genomes. Important functional similarities include (1) the presence of a capsid-bound RNA-dependent RNA polymerase, which produces single-stranded mRNA transcripts from the dsRNA genome and regenerates the dsRNA genome from single-stranded RNA templates; (2) in viruses infecting eukaryotic hosts, the presence of all the enzymatic activities needed to generate the 5' cap required by the eukaryotic translation machinery; (3) the high degree of structural order present in the packaged genome, suggesting the requirement for organization in the viral core; (4) the role of the innermost capsid protein as a scaffold on which the core components of the transcription apparatus are assembled; and (5) the release of nascent mRNA transcripts through channels at the icosahedral vertices. The process of genome transcription in dsRNA viruses will become better understood as structural studies progress to higher resolution and as more viruses become amenable to study using site-directed mutagenesis coupled with viral reconstitution to generate recombinant particles having precise functional and structural changes. Future studies will dissect important intermolecular interactions required for efficient mRNA synthesis and will shed further light on the reasons for which the viral core must be structurally intact in order for transcription to occur efficiently. Structural studies of the capping enzymes at atomic resolution will reveal how multiple enzyme activities reside within a single polypeptide and how they act in concert to synthesize the 5' cap on the end of each mature transcript. Pe...

show abstract

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Cited by 83 publications

References 35 publications

Self-Assembly of the Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses

Self-Assembly of the Recombinant Capsid Protein of a Bovine Norovirus (BoNV) into Virus-Like Particles and Evaluation of Cross-Reactivity of BoNV with Human Noroviruses

Strategies for manipulating the relative concentration of recombinant rotavirus structural proteins during simultaneous production by insect cells

Mechanism of genome transcription in segmented dsRNA viruses

Contact Info

Product

Resources

About