scholarly journals Feline Calicivirus VP2 Is Essential for the Production of Infectious Virions

2005 ◽  
Vol 79 (7) ◽  
pp. 4012-4024 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Gaël Belliot ◽  
Kyeong-Ok Chang ◽  
Oge Onwudiwe ◽  
Kim Y. Green
Keyword(s):  
Cdna Clone ◽  
Infectious Virus ◽  
Point Mutations ◽  
Eukaryotic Expression ◽  
Feline Calicivirus ◽  
Structural Protein ◽  
Reading Frame ◽  
Virus Particles ◽  
Minor Structural Protein ◽  
Productive Replication

ABSTRACT The third open reading frame (ORF3) located at the 3′ end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5′ end and small deletions in the 3′ end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3′ end.

scholarly journals Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells

2002 ◽  
Vol 76 (17) ◽  
pp. 8582-8595 ◽  
Author(s):  
Kim Y. Green ◽  
Aaron Mory ◽  
Mark H. Fogg ◽  
Andrea Weisberg ◽  
Gaël Belliot ◽  
...  
Keyword(s):  
Feline Calicivirus ◽  
Structural Protein ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Rna Molecules ◽  
Minor Structural Protein ◽  
Infected Cells ◽  
Positive Strand Rna ◽  
A Minor

ABSTRACT A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative “3A-like” protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.

scholarly journals Retroviral Env Glycoprotein Trafficking and Incorporation into Virions

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Tsutomu Murakami
Keyword(s):  
Infectious Virus ◽  
Structural Data ◽  
Host Factors ◽  
Structural Protein ◽  
Gag Protein ◽  
Virus Particles ◽  
Virion Incorporation ◽  
Actual Mechanism ◽  
Env Protein ◽  
Env Glycoprotein

Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

scholarly journals Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

2000 ◽  
Vol 74 (14) ◽  
pp. 6581-6591 ◽  
Author(s):  
Pamela J. Glass ◽  
Laura J. White ◽  
Judith M. Ball ◽  
Isabelle Leparc-Goffart ◽  
Michele E. Hardy ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Insect Cells ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Structural Protein ◽  
Coding Sequences ◽  
Reading Frame ◽  
Single Protein ◽  
Minor Structural Protein ◽  
A Minor

ABSTRACT Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

scholarly journals Genetic Requirements for the Function of the Archaeal Virus SSV1 in Sulfolobus solfataricus: Construction and Testing of Viral Shuttle Vectors

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1397-1405
Author(s):  
Kenneth M Stedman ◽  
Christa Schleper ◽  
Evelyn Rumpf ◽  
Wolfram Zillig
Keyword(s):  
Infectious Virus ◽  
Shuttle Vector ◽  
Sulfolobus Solfataricus ◽  
Open Reading Frames ◽  
Archaeal Virus ◽  
Reading Frame ◽  
Selection Technique ◽  
Virus Particles ◽  
E Coli ◽  
Thermophilic Archaeon

Abstract Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15.5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.

scholarly journals Isolation of Viable SARS-CoV-2 Virus from Feces of an Immunocompromised Patient Suggesting a Possible Fecal Mode of Transmission

2021 ◽  
Vol 10 (12) ◽  
pp. 2696
Author(s):  
Julie Dergham ◽  
Jeremy Delerce ◽  
Marielle Bedotto ◽  
Bernard La Scola ◽  
Valérie Moal
Keyword(s):  
Kidney Transplantation ◽  
Infectious Virus ◽  
Immunocompromised Patient ◽  
Kidney Transplant Recipient ◽  
Enteric Virus ◽  
Rt Pcr ◽  
Virus Particles ◽  
Severe Diarrhea ◽  
Mode Of Transmission ◽  
Compromised Patients

(1) Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) excretion in stools is well documented by RT-PCR, but evidences that stools contain infectious particles are scarce. (2) Methods: After observing a Corona Virus 2019 Disease (COVID-19) epidemic cluster associated with a ruptured sewage pipe, we search for such a viable SARS-CoV-2 particle in stool by inoculating 106 samples from 46 patients. (3) Results: We successfully obtained two isolates from a unique patient with kidney transplantation under immunosuppressive therapy who was admitted for severe diarrhea. (4) Conclusions: This report emphasizes that SARS-CoV-2 is an enteric virus, and infectious virus particles can be isolated from the stool of immune-compromised patients like, in our case, kidney transplant recipient. Immune-compromised patients are likely to have massive multiplication of the virus in the gastrointestinal tract and this report suggests possible fecal transmission of SARS-CoV-2.

scholarly journals RG1-VLP and Other L2-Based, Broad-Spectrum HPV Vaccine Candidates

2021 ◽  
Vol 10 (5) ◽  
pp. 1044
Author(s):  
Bettina Huber ◽  
Joshua Weiyuan Wang ◽  
Richard B. S. Roden ◽  
Reinhard Kirnbauer
Keyword(s):  
Small Animal ◽  
Good Manufacturing Practice ◽  
Special Focus ◽  
T Cell Epitopes ◽  
Hpv Vaccines ◽  
Structural Protein ◽  
Vaccine Candidates ◽  
Protective Antibodies ◽  
Minor Structural Protein ◽  
Hpv Types

Licensed human papillomavirus (HPV) vaccines contain virus-like particles (VLPs) self-assembled from L1 major-capsid proteins that are remarkably effective prophylactic immunogens. However, the induced type-restricted immune response limits coverage to the included vaccine types, and costly multiplex formulations, restrictive storage and distribution conditions drive the need for next generation HPV vaccines. Vaccine candidates based upon the minor structural protein L2 are particularly promising because conserved N-terminal epitopes induce broadly cross-type neutralizing and protective antibodies. Several strategies to increase the immunological potency of such epitopes are being investigated, including concatemeric multimers, fusion to toll-like receptors ligands or T cell epitopes, as well as immunodominant presentation by different nanoparticle or VLP structures. Several promising L2-based vaccine candidates have reached or will soon enter first-in-man clinical studies. RG1-VLP present the HPV16L2 amino-acid 17–36 conserved neutralization epitope “RG1” repetitively and closely spaced on an immunodominant surface loop of HPV16 L1-VLP and small animal immunizations provide cross-protection against challenge with all medically-significant high-risk and several low-risk HPV types. With a successful current good manufacturing practice (cGMP) campaign and this promising breadth of activity, even encompassing cross-neutralization of several cutaneous HPV types, RG1-VLP are ready for a first-in-human clinical study. This review aims to provide a general overview of these candidates with a special focus on the RG1-VLP vaccine and its road to the clinic.

scholarly journals Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies

2008 ◽  
Vol 82 (16) ◽  
pp. 7897-7904 ◽  
Author(s):  
Oihane Simón ◽  
Trevor Williams ◽  
Aaron C. Asensio ◽  
Sarhay Ros ◽  
Andrea Gaya ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Gene Deletion ◽  
Particle Content ◽  
Reading Frame ◽  
Virus Particles ◽  
Multiple Nucleopolyhedrovirus ◽  
Glycosylation Sites ◽  
Occlusion Bodies ◽  
Viral Factor ◽  
Positive Controls

ABSTRACT The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB.

scholarly journals STRUCTURE OF TYPE 5 ADENOVIRUS

1963 ◽  
Vol 118 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Wesley C. Wilcox ◽  
Harold S. Ginsberg
Keyword(s):  
Virus Particle ◽  
Density Gradient ◽  
Infectious Virus ◽  
Relative Proportion ◽  
Specific Antigen ◽  
Equilibrium Density ◽  
Virus Particles ◽  
Infected Cells ◽  
Specific Antigens ◽  
Virus Antigens

Type 5 adenovirus was purified by fluorocarbon (freon 113) treatment followed by banding in a CsCl equilibrium density gradient. This method permitted separation of virus from normal host cell materials and virus-specific soluble antigens. Virus banded in CsCl with a mean bouyant density of 1.3349 gm/cm3. The three virus-specific soluble antigens (group- and type-specific antigens and toxin) banded together with a mean bouyant density of 1.2832 gm/cm3. The group-specific antigen was the predominant antigen of the purified virus particle, whereas the group- and type-specific antigens were present in equal titers in the antigen band. Infectious virus particles were inactivated by prolonged dialysis at pH 10.5. Centrifugation of inactivated virus preparations in a CsCl equilibrium density gradient resulted in separation of virus DNA from specific antigen: the antigens banded with a mean bouyant density of 1.2832 gm/cm3 and the DNA sedimented to the bottom of the tube. The predominant antigen derived from purified virus particles was the group-specific antigen and it was in the same relative proportion to the type-specific antigen as measured in intact particles. The antigens derived from disrupted virus were immunologically identical with the soluble virus antigens present in infected cells.

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