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The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions

The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions | Virology News | Scoop.it

The strength of attraction between capsid proteins (CPs) of cowpea chlorotic mottle virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and single-stranded RNA as a function of the strength of CP–CP and CP–RNA attractions. Using the techniques of velocity sedimentation and electron microscopy, we find that the successful assembly of nuclease-resistant virus-like particles (VLPs) depends delicately on the strength of CP–CP attraction relative to CP–RNA attraction. If the attractions are too weak, the capsid cannot form; if they are too strong, the assembly suffers from kinetic traps. Separating the process into two steps—by first turning on CP–RNA attraction and then turning on CP–CP attraction—allows for the assembly of well-formed VLPs under a wide range of attraction strengths. These observations establish a protocol for the efficient in vitro assembly of CCMV VLPs and suggest potential strategies that the virus may employ in vivo.

 
Ed Rybicki's insight:

I do love it when a paper is published that could have been done pretty much any time in the last 40 years - and with one of my favourite viruses, that I played with a LOT back there before 1980.

Ultracentrifugation, pH meters, ionic strength determinations, EM...all tried and true, and used 40+ years ago. OK, they also used cloned BMV RNA 1 cDNA, and did 3-D image reconstruction from EMs, but hey, they needn't have done that!  Nice, straightforward physicochemical studies, on a well-characterised virus, with good, simple conclusions. 

Namely, that assembly of the virus is NOT just a simple mix-CP-and-RNA-and-it-will-happen, but that it depends upon both pH, for modulating ionic interactions,and ionic strength for modulating ionic interactions AND the "hydrophobic effect", as we used to know it.

While their conclusions are relevant for assembly of heat- and nuclease-resistant nano particles in vitro, I wonder if they are physiologically relevant: if "correct" assembly depends upon first, turning on CP-RNA attraction (ionic strength shift), and second, turning on CP-CP attraction (pH shift) - where in the cell does that happen?

In their own words, "It is generally accepted that the cytoplasm of plant cells is maintained near neutral pH with ionic strength of approximately 0.1 M. Our in vitro results show that these conditions are insufficient for nucleocapsid formation in the absence of cellular host factors."

Yeeee-ee-eesssss...precisely. What happens in the cell? The answer could lie in the one thing they don't report, but that some of the heroes of my distant youth - people like JB Bancroft and Thom Hohn, both quoted (from 1970 and 1969 respectively) in this paper, DID do. Namely, investigate what happens at different CP and RNA concentrations, at constant pH and ionic strength.

You see, it was shown 30+ years ago - and I have been lecturing on it since then - that CP and RNA for viruses like BMV / CCMV and MS2 form different complexes with their cognate partners at different molecular ratios. That is, at low CP:RNA ratios, a high-affinity complex is formed, which is basically a ribonucleoprotein complex without structure. Increasing the CP:RNA ratio for both MS2 and CCMV, as I recall (maybe Dick Verduin was involved with CCMV), results in further lower-affinity association of CP with both RNA and already-bound CP - which acts as a nucleation complex - to result in full capsid assembly.

I note that the process in both cases was shown to be specific, for low CP:RNA ratios: that is, it was cognate CP and RNA doing the high affinity nucleation complex formation.

And these guys deliberately used a heterologous RNA...albeit one from a related virus, but still: what would have happened if they'd used CCMV RNA?

Still - great paper, taking me back to when I wrote an essay on "Assembly of Spherical Plant Viruses" in my Honours year in 1977, quoting quite a few of the same references these folk did. Ah, simpler times...B-)

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Nanoparticle Flu Vaccine Provides Protection against Six Viral Strains

From BioPortfolio: Researchers developed and showed that a novel nanoparticle vaccine that combines two major influenza proteins is effective in providing broad, long-lasting prot...
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The impact of repeated vaccination using 10-year vaccination history on protection against influenza in older adults

Introduction
Annual influenza vaccination is recommended for older adults, but evidence regarding the impact of repeated vaccination has been inconclusive. Aim We investigated vaccine effectiveness (VE) against laboratory-confirmed influenza and the impact of repeated vaccination over 10 previous...
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Hot rocks offer clues to life on 'Snowball Earth'

Hot rocks offer clues to life on 'Snowball Earth' | Virology News | Scoop.it
Scientists braved the heat to answer a question about oxygen and "Snowball Earth," a period when the oceans virtually froze from the poles to the equator.
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Australian researchers developing vaccine for peanut allergies

Australian researchers developing vaccine for peanut allergies | Virology News | Scoop.it
Researchers from the University of South Australia are close to developing a life-changing vaccine for deadly peanut allergies.
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Osivax Enters Agreement with U.S. National Institutes of Health to Test Universal Flu Vaccine Candidate

Osivax Enters Agreement with U.S. National Institutes of Health to Test Universal Flu Vaccine Candidate | Virology News | Scoop.it
Lyon, France – December 5, 2019  – Osivax announced today that it has entered into an agreement with the United States’ National Institute of Allergy and Infectious Diseases (NIAID), part of...
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Probable mass graves uncovered in Queensland town of Cherbourg decimated by Spanish influenza 

Probable mass graves uncovered in Queensland town of Cherbourg decimated by Spanish influenza  | Virology News | Scoop.it
An Aboriginal elder in Queensland is working with a team of researchers to find mass graves — the final resting place of 90 locals who died in the space of three weeks during one of the world's worst outbreaks of Spanish influenza.
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xkcd: Flu Shot

xkcd: Flu Shot | Virology News | Scoop.it
Flu Shot(alt-text) "Wait, how often are you getting bitten by snakes? And why are you boiling water?" "Dunno, the CDC people keep showing up with complicated questions about the 'history of the property' and 'possible curses' but I kinda tune them out. At least one of them offered me the flu shot." |< < ? > >| You can get it here: Amazon, B&N, IndieBound, Apple, Audible, Target Archive Store What If? About
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Grandparents' vaccines – what's recommended before they hold newborns

Grandparents' vaccines – what's recommended before they hold newborns | Virology News | Scoop.it
It's that time of year when we should think about the grandparents' vaccines before they meet and hold newborns.And also these vaccines protect them...
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Vaccine: Flu victim recovering from three-month coma

Vaccine: Flu victim recovering from three-month coma | Virology News | Scoop.it
A man with asthma who caught the flu ended up in a coma after declining a flu vaccine.
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Cancer's 'addiction' could be a way to defeat it

Cancer's 'addiction' could be a way to defeat it | Virology News | Scoop.it
Scientists call some cancers "Wnt addicted" because they can't live without it. New research with gastric cancer organoids suggests a new treatment strategy.
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To cope with anxiety, use 'safety signals'

To cope with anxiety, use 'safety signals' | Virology News | Scoop.it
"A safety signal could be a musical piece, a person, or even an item like a stuffed animal that represents the absence of threat."...
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Rats replay memories forward and backwards to make decisions

Rats replay memories forward and backwards to make decisions | Virology News | Scoop.it
Watching the way rat brains replay their memories forward and backward while making a decision let researchers predict what it would decide.
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KISS cancel entire Australian tour due to Paul Stanley illness

KISS cancel entire Australian tour due to Paul Stanley illness | Virology News | Scoop.it
KISS have been forced to cancel their entire Australian tour as Paul Stanley continues to battle a bad bout of influenza.
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Don't know if that's a bad or a good thing...?
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Meissa Vaccines Receives U.S. FDA Fast Track Designation for Respiratory Syncytial Virus Vaccine, MV-012-968

Meissa Vaccines Receives U.S. FDA Fast Track Designation for Respiratory Syncytial Virus Vaccine, MV-012-968 | Virology News | Scoop.it
Meissa Vaccines Receives U.S.FDA Fast Track Designation for Respiratory Syncytial Virus Vaccine, MV-012-968...
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Self-assembly into virus-like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

PCV3 capsid protein (Cap) is an important antigen for diagnosis and vaccine development. To achieve high-level expression of recombinant PCV3 Cap in Escherichia coli (E. coli), the gene of wild-type entire Cap (wt-eCap) was amplified from clinical samples, and three optimized entire Cap (opti-eCap) and one optimized Cap deleted nuclear location signal (NLS) (opti-dCap) gene fragments encoding the same amino acid sequence with wt-eCap were synthesized based on the codon bias of E. coli. Those gene fragments were inserted into the pET30a expression vector. One recombinant strain with the highest expressed soluble eCap from four entire Cap (one wt-eCap and three opti-eCap) and one recombinant strain expressed opti-dCap were selected for further purification. The purified eCap and dCap were identified by transmission electron microscopy (TEM), a large number of round hollow particles with a diameter of 10 nm virus-like particles (VLPs) were observed in eCap, whereas irregular aggregation of proteins observed in dCap. After formation the VLPs were applied as a coating antigen to establish an indirect ELISA (I-ELISA) for detection of PCV3-specific antibody in swine serum. 373 clinical swine serum samples from China collected in 2019 were tested utilizing the VLP-based I-ELISA method under optimized conditions. To the best of our knowledge, this is the first report of self-assembly into VLPs of PCV3 recombinant Cap. Our results demonstrated that the VLP-based I-ELISA will be a valuable tool for detecting the presence of PCV3 antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes.

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Giant ice planet orbits hot star 1/4 its size

Giant ice planet orbits hot star 1/4 its size | Virology News | Scoop.it
New evidence points to an giant, icy planet orbiting a white dwarf star that's slowing evaporating it.
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Preparing for the Next Pandemic — The WHO’s Global Influenza Strategy

Preparing for the Next Pandemic — The WHO’s Global Influenza Strategy | Virology News | Scoop.it
Preparing for the Next Pandemic Given the ongoing threat posed by influenza, the WHO earlier this year released its Global Influenza Strategy 2019–2030. Although the strategy is a welcome step, add...
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Half a million Ebola vaccine doses to be stockpiled for emergency use 

Half a million Ebola vaccine doses to be stockpiled for emergency use  | Virology News | Scoop.it
- A stockpile of 500,000 doses of Ebola vaccine for emergency use in outbreaks o...
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U.S. flu season arrives early and it could be an especially bad one, experts warn

U.S. flu season arrives early and it could be an especially bad one, experts warn | Virology News | Scoop.it
There’s a good chance flu season could peak much earlier than normal in the U.S. this winter, health officials say.
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Antibody May Make Effective Norovirus Vaccine 

Antibody May Make Effective Norovirus Vaccine  | Virology News | Scoop.it
Norovirus Antibody May Make Effective “Stomach Flu” Vaccine  Genetic Engineering & Biotechnology News...
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NASA's Parker probe reveals new solar oddities

NASA's Parker probe reveals new solar oddities | Virology News | Scoop.it
The first observations from NASA's Parker Solar Probe "will fundamentally change our understanding of the sun," researchers say.
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3 theories why asteroid Bennu is spewing bits

3 theories why asteroid Bennu is spewing bits | Virology News | Scoop.it
"Among Bennu's many surprises, the particle ejections sparked our curiosity, and we've spent the last several months investigating this mystery."...
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New calf pneumonia vaccine to protect calves as early as 1 week

New calf pneumonia vaccine to protect calves as early as 1 week | Virology News | Scoop.it
A new early life intranasal vaccine will allow UK cattle farmers can now protect dairy and beef calves against the key causes of viral pneumonia from as young as one week of age.
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China reports two human H9N2 avian flu cases

China reports two human H9N2 avian flu cases | Virology News | Scoop.it
By NewsDesk   @bactiman63 Taiwan health officials reported today (computer translated) on two human H9N2 avian influenza on Mainland China. The cases, from November 12th and 22nd, are the first reported in China this season.
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Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

BACKGROUND: Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G710 to A710) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. RESULTS: Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID50), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. CONCLUSIONS: These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

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