Physics of Viral Infectivity: Energetics of Genome Ejection

Meerim Jeembaeva

Physics of Viral Infectivity: Energetics of Genome Ejection

Meerim Jeembaeva

Biokemi och Strukturbiologi

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

Sammanfattning

Popular Abstract in Swedish
Virala infektioner är och har varit ett stort hälsoproblem för mänskligheten. Många av de sjukdomar som förorsakas av virus är obotliga och spridningen är oftast snabb (ett exempel är HIV). Förutom att direkt förorsaka olika sjukdomstillstånd har virus också påvisats vara orsak till olika former av cancer.
Under de senaste åren har dock forskare försökt att börja använda virus för att bekämpa virussjukdomar och andra obotliga genetisk betingade sjukdomar. Strategin har varit att använda virus för att leverera ”friskt” icke viralt DNA in till de sjuka cellerna och därmed bota dessa. Denna metod fick namnet genterapi. Trots att de första försöken gjordes redan för 15 år sedan så fungerar inte metoden ännu idag. En av anledningarna till att man inte har lyckats att förstå och kontrollera beteendet hos olika virus är att väldigt få eller nästan inga studier har gjorts där man undersöker de fysikalisk-kemiska aspekterna kring virusets reproduktion.
Eftersom virologer och strukturbiologer redan skaffat en hel del kunskap om virus struktur och dess biokemiska funktioner är tiden nu mogen för att även undersöka de fundamentala fysikalisk-kemiska egenskaperna hos virus för att få kontroll över dessa och därmed bättre kunna försvara oss mot olika virusangrepp.
Virus är bland de enklaste biologiska objekt i vår omgivning. Vanligen består de av en behållare av proteinmolekyler, en så kallad viruskapsel, som omsluter virusets DNA (eller RNA). Därmed utgör virus en relativt enkel fysikalisk modell som kan beskrivas med dagens analytiska och numeriska teorier från fysiken. Liksom i andra organismer är det virusets DNA molekyl som bär den information som behövs för att reproducera viruset. När viruspartikeln sätts ihop i en infekterad cell, så måste dess genommolekyl (arvsmassa), som kan vara upp till 15 mikrometer lång, tryckas ihop i en viruskapsel med en volym som är 150 gånger mindre i diameter. Vi har kunnat visa att det ihoptryckta DNA i detta bakteriella virus måste utöva ett mycket högt tryck inuti kapseln. Vi har kunnat bestämma detta tryck som visade sig vara så högt som 50 atmosfärer i visa virus, vilket motsvarar trycket på 500 m djup i havet. Detta experiment är den första mätningen av tryck och kraft i virus, vilket ger en förklaring till drivkraften för injektion av DNA från bakteriella virus under infektionen av en bakteriecell.
Baserat på dessa framgångsrika experiment, och med stöd av teori, har vi genomfört en bred studie av virus där energi, tryck och kraft hos viralt DNA inne i en viruskapsel har bestämts som funktion av DNAs längd, det osmotiska trycket samt salthalt i den omgivande lösningen. Våra studier har även omfattat en kalorimetrisk undersökning av energin associerad med utsprutningen av DNA från en viruskapsel.
Sammanfattningsvis ger detta forskningsarbete en bred beskrivning av den fysikaliska mekanismen som styr virus infektionen och ger en möjlighet att kunna kontrollera och även stoppa den. Dessutom kommer studierna att öppna porten till strategier för att omvandla virus till läkemedelsbärare som kan användas för att bota hittills obotliga sjukdomstillstånd.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution	Biokemi och Strukturbiologi
Handledare	Evilevitch, Alex, handledare
Tilldelningsdatum	2011 mars 15
Förlag	Department of Chemistry, Lund University
ISBN (tryckt)	978-91-7422-264-7
Status	Published - 2011

Bibliografisk information

Defence details

Date: 2011-03-15
Time: 13:15
Place: Lecture Hall B, Center for Chemistry and Chemical Engineering

External reviewer(s)

Name: Bamford, Dennis
Title: Professor
Affiliation: Finnish Centre of Excellence in Virus Research, University of Helsinki, Helsinki, Finland

---

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Biologiska vetenskaper

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N2 - All viruses that infect bacteria, plant, or animal cells involve a genome (RNA or DNA) that is encapsidated by a rigid protein shell. After delivery of the viral genome into the host cell, new capsid proteins, which are encoded by viral DNA or RNA, are expressed and self-assembled into new viral capsids. The main objective of my research was to study the key physical factors of genome ejection that control the viral life cycle. One of the main steps in the viral life cycle is genome ejection, which is considered a physical process. It has recently been shown that the ejection of the viral genome from the phage capsid is driven by internal pressure reaching tens of atmospheres. This pressure is partially responsible for the delivery of the viral genome into the host cell, thus making it central in the infection process. In this doctoral thesis, direct measurements of the energy associated with genome ejection are presented. The viral capsid is “opened” by the LamB receptor protein in vitro, and ejection is measured using microcalorimetry. The energy of genome ejection from the viral capsid is obtained as a function of the relative packaging density of the viral genome. A DNA phase transition was observed by measuring the ejection enthalpy as a function of temperature. Recent in vitro experiments have shown that DNA ejection from the phage can be restricted by the surrounding osmotic pressure when ejected DNA is digested by DNase I during the course of ejection. The most important finding of this work was that the ejection of an intact genome (i.e. undigested) in a crowded environment is enhanced or even completed by the pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that, in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it, while in vitro ejection is extremely dependent on the pressure within the virus capsid. The effect of internal pressure on the infection of a bacterial cell is unknown. A microfluidic technique was employed to monitor individual cells and determine the distribution of lysis due to infection in relation to the capsid pressure. The probability of lysis was found to decrease markedly with decreasing capsid pressure.

AB - All viruses that infect bacteria, plant, or animal cells involve a genome (RNA or DNA) that is encapsidated by a rigid protein shell. After delivery of the viral genome into the host cell, new capsid proteins, which are encoded by viral DNA or RNA, are expressed and self-assembled into new viral capsids. The main objective of my research was to study the key physical factors of genome ejection that control the viral life cycle. One of the main steps in the viral life cycle is genome ejection, which is considered a physical process. It has recently been shown that the ejection of the viral genome from the phage capsid is driven by internal pressure reaching tens of atmospheres. This pressure is partially responsible for the delivery of the viral genome into the host cell, thus making it central in the infection process. In this doctoral thesis, direct measurements of the energy associated with genome ejection are presented. The viral capsid is “opened” by the LamB receptor protein in vitro, and ejection is measured using microcalorimetry. The energy of genome ejection from the viral capsid is obtained as a function of the relative packaging density of the viral genome. A DNA phase transition was observed by measuring the ejection enthalpy as a function of temperature. Recent in vitro experiments have shown that DNA ejection from the phage can be restricted by the surrounding osmotic pressure when ejected DNA is digested by DNase I during the course of ejection. The most important finding of this work was that the ejection of an intact genome (i.e. undigested) in a crowded environment is enhanced or even completed by the pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that, in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it, while in vitro ejection is extremely dependent on the pressure within the virus capsid. The effect of internal pressure on the infection of a bacterial cell is unknown. A microfluidic technique was employed to monitor individual cells and determine the distribution of lysis due to infection in relation to the capsid pressure. The probability of lysis was found to decrease markedly with decreasing capsid pressure.

KW - microfluidics.

KW - differential scanning calorimetry

KW - capsid

KW - Key words: phage

KW - osmotic suppression

KW - DNA ejection

KW - isothermal titration calorimetry

KW - enthalpy

M3 - Doctoral Thesis (compilation)

SN - 978-91-7422-264-7

PB - Department of Chemistry, Lund University

ER -

Physics of Viral Infectivity: Energetics of Genome Ejection

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