Replication of DNA Virus Genomes

YORAPERMATADEWI

REPLICATIONOFDNAVIRUSGENOMES

YORAPERMATADEWI,MIKROBIOLOGIFKUI

1.NUCLEARIMPORT

• MOST DNA VIRUSES REPLICATE IN THE NUCLEUS, THEREFORE,THEIR VIRAL GENOMEMUST ENTER THE NUCLEUS OF THE HOSTCELL.

• ALTHOUGH THERE ARE NUMEROUS BENEFITS, ENTRY INTO THENUCLEUS ALSO POSES A SERIOUS CHALLENGE FOR THESEVIRUSES,SINCETHENUCLEARENVELOPE(NE)ACTSASABARRIERBETWEEN THE CYTOPLASMAND THENUCLEUS, AND TRANSPORTOF MOLECULES INTO AND OUT OF THE NUCLEUS IS TIGHTLYREGULATED.

• TO ENTER THE NUCLEUS, THEY CAN MAKE USE OF THE NPC(NUCLEAR PORE COMPLEX) FOR TRANSPORT OF THE GENOMEANDACCESSORYPROTEINSINTOTHENUCLEOPLASM.


• TWO GENERAL MECHANISMS HAVE BEEN DESCRIBED FORNUCLEARIMPORT:• PASSIVEDIFFUSION• FACILITATEDTRANSLOCATION

• PASSIVEDIFFUSIONISFORIONSANDMOLECULESSMALLERTHAN9nmINDIAMETERORPROTEINSSMALLERTHAN40kDA

•  FACILITATED NUCLEAR IMPORT CAN ACCOMMODATE THETRANSPORTOFMOLECULESWITHDIAMETERSOFUPTO39nm.


• THE FACILITATED NUCLEAR IMPORT MECHANISM REQUIRES ASIGNAL RESIDING ON THE IMPORTED MOLECULE (OR CARGO),AND CYTOPLASMIC RECEPTORS (CALLED NUCLEAR IMPORTRECEPTORS, IMPORTINS, OR KARYOPHERINS) THAT RECOGNIZETHE SIGNAL ANDMEDIATE THE TRANSLOCATION OF THE CARGOTHROUGHTHENPC.

• ALTHOUGHTHEREAREMANYTYPESOFNUCLEARLOCALIZATIONSEQUENCES (NLSs), THE FIRST IDENTIFIED AND MOST STUDIEDSIGNAL CONSISTS OF ONE OR TWO SHORT STRETCHES OF BASICAMINOACIDS,CALLEDTHECLASSICALNLS(CNLS).


CLASSICALPROTEINIMPORTPATHWAY(CLASSICALNLS[CNLS])

Flint,2015;p.152


ARRANGEMENTOFNPCsINNE

Albert,2015;p.651


• KEY PLAYERS INVOLVED IN THIS PROCESS OFNUCLEAR IMPORTARE:• THENPC,• NUCLEAR LOCALIZATION SEQUENCES (NLSs) ON THETRANSPORTEDPROTEINS,• NUCLEARTRANSPORTRECEPTORS(NTRs)THATRECOGNIZETHENLSsINTHETRANSPORTEDPROTEINS,AND•  THESMALLRas-likeGTPaseRan.

• BECAUSE THE SIZE AND STRUCTURE OF VIRUSES VARYENORMOUSLY AND BECAUSE THERE ARE SEVERAL NUCLEARIMPORT PATHWAYS, EACH VIRUS HAS EVOLVED A UNIQUESTRATEGYTODELIVERITSGENOMEINTOTHENUCLEUS.

YORAPERMATADEWI,MIKROBIOLOGIFKUI Cohen,2011

YORAPERMATADEWI,MIKROBIOLOGIFKUI Fay&Fante,2015


NUCLEARENTRYOFENVELOPEDDNAVIRUSES

YORAPERMATADEWI,MIKROBIOLOGIFKUI Wikipedia


NUCLEARENTRYOFNON-ENVELOPEDDNAVIRUSES


LIGHTANDHEAVYCHAINOFKINESIN-1

Wikipedia


DNAVIRUSGENOMESTRATEGIES• DNA VIRUSES WITH SMALL GENOMES SUCH AS POLYOMA-,PAPILLOMA-, AND PARVOVIRUSES USE HOST-CELL ENZYMES FORTRANSCRIPTIONANDREPLICATION.• DNA VIRUSES WITH INTERMEDIATE-SIZE GENOMES (UP TO 35 KB)SUCH AS ADENOVIRUSES, ENCODE MUCH OF THEIR DNAREPLICATION MACHINERY INCLUDING A DNA POLYMERASE,TERMINAL PROTEIN AND ssDNA BINDING PROTEIN, BUT THEYEMPLOY CELLULAR RNA POLYMERASE I I AND I I I FORTRANSCRIPTION.• DNAVIRUSESWITHLARGERGENOMES(150TO350KB),SUCHASTHEHERPESVIRUSESANDPOXVIRUSES,ENCODEDNAPOLYMERASESANDBINDINGPROTEINS.• HEPADNAVIRUSES BUCK THIS GENERAL TREND IN THAT THEY ARESMALL GENOMES (3 KB) BUT ENCODE THE DNA POLYMERASE/REVERSE TRANSCRIPTASE (RT) THAT EXECUTES THEIR UNIQUEMECHANISMOFDNAREPLICATIONVIAANssRNAINTERMEDIATE.

YORAPERMATADEWI,MIKROBIOLOGIFKUI Flint,2015



• REPLICATIONOFALLDNA,FROMTHEGENOMEOFTHESIMPLESTVIRUS TO THAT OF THE MOST COMPLEX VERTEBRATE CELL,FOLLOWSASETOFUNIVERSALRULES:•  DNA IS ALWAYS SYNTHESIZED BY TEMPLATE-DIRECTED, STEPWISEINCORPORATION OF DEOXYNUCLEOSIDEMONOPHOSPHATES (dNMPs)FROM DEOXYNUCLEOSIDE TRIPHOSPHATE (dNTP) SUBSTRATES INTOTHE3 ︎-OHENDOFTHEGROWINGDNACHAIN;•  EACH PARENTAL STRANDOF A DUPLEX DNA TEMPLATE IS COPIED BYBASEPAIRINGTOPRODUCETWODAUGHTERMOLECULESIDENTICALTOONE ANOTHER AND TO THEIR PARENT (SEMI-CONSERVATIVEREPLICATION);•  REPLICATION OF DNA BEGINS AND ENDS AT SPECIFIC SITES IN THETEMPLATE,TERMEDORIGINSANDTERMINI,RESPECTIVELY;AND•  DNASYNTHESISISCATALYZEDBYDNA-DEPENDENTDNAPOLYMERASES,BUTMANY ACCESSORY PROTEINS ARE REQUIRED FOR INITIATIONORELONGATION. INCONTRASTTOALLDNA-DEPENDENT,ANDMANYRNADEPENDENT,RNAPOLYMERASES,NODNAPOLYMERASECAN INITIATETEMPLATE-DIRECTEDDNASYNTHESISDENOVO.ALLREQUIREAPRIMERWITHAFREE3 ︎-OHENDTOWHICHdNMPsCOMPLEMENTARYTOTHOSEOFTHETEMPLATESTRANDAREADDED.



TWOMECHANISMOFdsDNASYNTHESIS

Flint,2015

COPYING OF BOTH STRANDS OF ADOUBLE-STRANDEDDNATEMPLATEATAREPLICATIONFORK

COPYING OF ONLY ONE STRANDWH I L E I T S C OMP L EMEN T I SDISPLACED


STRANDDISPLACEMENTOFADENOVIRUS

REQUIREMENTS:

u  PRETERMINALPROTEIN(pTP)u  CARRIESACYTIDINENUCLEOTIDE

THATPROVIDESTHEPRIMER

u  ASSOCIATEDWITHDNAPOLYMERASE

u  DNAPOLYMERASEAdV(Pol)

u  DNABINDINGPROTEIN(DBP)

Krebs,Goldstein,Kilpatrick,2011


PROTEINSINVOLVEDINAdDNAREPLICATION

Hay,1996


S T R A N D D I S P L A C E M E N TMECHANISMTHEREFORERESULTSI N S E M I C O N S E R V A T I V EREPLICATION EVEN THOUGH THETWO PARENTAL STRANDS OFVIRAL DNA ARE NOT COPIED ATTHESAMEREPLICATIONFORK.

Flint,2015;p.280


InvertedTerminalRepeat(ITR)• REQUIREDFOREFFICIENTMULTLIPICATIONOFAdVGENOME.• THESE SEQUENCES GIVE ABILITY TO FORM A HAIRPIN OR PAN-HANDLE DUPLEX, WHICH CONTRIBUTES TO SELF-PRIMING THATALLOWSPRIMASE-INDEPENDENTSYNTHESISOFTHESECONDDNASTRAND.•  ITS ALSO SHOWN TO BE REQUIRED FOR BOTH INTEGRATION OFTHEADVDNAINTOTHEHOSTCELLGENOME.


2.REPLICATIONMECHANISMOFDNAVIRUSES

1.  THECIRCULARdsDNAGENOMESOFPOLYOMAVIRUSESANDPAPILLOMAVIRUSES REPLICATE BIDIRECTIONALLY FROM ASINGLEAT-RICHORIGINVIATHERNA-PRIMEDSYNTHESISOFCONTINUOUS LEADING STRANDS AND DISCONTINUOUSLAGGING STRANDS AT BOTH REPLICATION FORKS.CIRCULARITY OF THE GENOME ASIDE, THE REACTIONS ATTHE REPLICATION FORKS CLOSELY RESEMBLE HOW THEHOSTCHROMOSOMEISREPLICATED.


SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFPOLYOMAVIRIDAEANDPAPILLOMAVIRIDAE

Field,Knipe,Howley,2013


2. IN STARK CONTRAST, THE LINEAR dsDNA GENOME OFADENOVIRUSES IS REPLICATED BY A PROTEIN-PRIMEDSYNTHESIS OF ONLY THE LEADING STRAND, RESULTING INDISPLACEMENTOF ssDNAFROMEACHENDOFTHEPARENTALDUPLEX.THETERMINIOFTHEDISPLACEDSTRANDSANNEALVIAINVERTEDTERMINALREPEATS,CREATINGDUPLEXPANHANDLESTRUCTURES THAT SERVE AS SECONDARY ORIGINS OFREPLICATION. THE PRIMER (PRETERMINAL PROTEIN) IS THEPRODUCTOFANEARLYGENE,ANDACOPYOFTHISPROTEINISCOVALENTLY BOUND TO THE 5′ END OF EACH OF THEDAUGHTERSTRANDS.


SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFADENOVIRIDAE



3. THE LINEAR dsDNA OF HERPESVIRUS GENOMES IS FIRSTCIRCULARIZED AND THEN REPLICATED FROM ONE OR MOREINTERNAL ORIGINS, MOST LIKELY BY AN RNA-PRIMEDMECHANISM THAT EVENTUALLY PRODUCES dsDNACONCATAMERS.PROGENYDNACANUNDERGOISOMERIZATIONBY HOMOLOGOUS RECOMBINATION BETWEEN INTERNAL ANDTERMINAL REPEATED SEQUENCES, AND UNIT LENGTHGENOMES ARE RESOLVED FROM THE CONCATAMERS DURINGPACKAGINGINTOVIRIONS.


SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFHERPESVIRIDAE



4.DESPITETHEIRDIFFERENTSTRUCTURESANDSIZES,POXVIRUSAND PARVOVIRUS GENOMES REPLICATE BY SIMILARMECHANISMS. THE CLOSE-ENDED DUPLEX POXVIRUS GENOME(ORTHECLOSED-ENDEDDUPLEXINTERMEDIATEINPARVOVIRUSREPLICATION) ISNICKEDNEAR ITS TERMINUS,ANDTHENEWLYGENERATED3’ENDSERVESTOPRIMEDNASYNTHESISUSINGTHECOMPLEMENTARY STRANDOF THEDUPLEXAS TEMPLATE. THISINITIAL SELF-PRIMING EVENT IS REPRODUCED BY PARTIALLYBASE-PAIRED HAIRPIN STRUCTURES LOCATED AT EACH END OFTHE DUPLEX GENOME, RESULTING IN SO CALLED “ROLLINGHAIRPIN” REPLICATION. FOR BOTH POXVIRUSES ANDPARVOVIRUSES,THEPRODUCT ISAdsDNACONCATAMERFROMWHICHUNITLENGTHGENOMESAREEXCISEDBYRESOLUTIONOFCONCATAMERJUNCTIONS.


SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFPOXVIRIDAE



SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFPARVOVIRIDAE



5. FINALLY, IN THE MOST TORTUOUS MECHANISM OF ALL,HEPADNAVIRUSES REPLICATE THEIR dsDNA GENOME BY AFULL-LENGTHPREGENOMICssRNATRANSCRIPTMADEBYRNAPOLYMERASE II. PREGENOMIC RNA IS THEN REVERSETRANSCRIBED BY THE VIRAL ENCODED DNA POLYMERASE/REVERSE TRANSCRIPTASE TO PRODUCE dsDNA PROGENY. INCONTRAST TO RETROVIRUSES, DNA INTEGRATION IS NOTREQUIRED FOR HEPADNAVIRUS REPLICATION, THE GENOMEBEINGMAINTAINEDASACIRCULAREPISOMEINTHENUCLEUSOFINFECTEDCELLS.


SIMPLIFIEDVIEWOFTHEREPLICATIONSCHEMEOFHEPADNAVIRIDAE



HEPADNAVIRUS

Flint,2015


PROCESSINGOFVIRALPRE-mRNA• TWOMODIFICATIONSIMPORTANTFOREFFICIENTTRANSLATION:q THEADDITIONOFM7GPPPN(7-METHYLGUANOSINERESIDUE)TOTHE5 ︎END(CAPPING)

q THE ADDITION OF MULTIPLE A NUCLEOTIDES TO THE 3 ︎ END(POLYADENYLATION)

• WHEN AN RNA IS PRODUCED IN THE NUCLEUS, ANOTHERCHEMICAL REARRANGEMENT, CALLED SPLICING, IS POSSIBLE.DURING SPLICING, SHORT BLOCKSOFNONCONTIGUOUS CODINGSEQUENCES (EXONS) ARE JOINED PRECISELY TO CREATE ACOMPLETE PROTEIN-CODING SEQUENCE FOR TRANSLATION,WHILETHEINTERVENINGSEQUENCES(INTRONS)AREDISCARDED.


RNAPROCESSING

Flint,2015


REFERENCE• FieldsB,KnipeDM,HowleyPM.Fieldsvirology.6ed.Philadelphia:LippincottWilliams&Wilkins;2013.

• Flint SJ. Principles of virology. 4 ed. Washington, DC: AmericanSocietyforMicrobiologyPress;2015.

• Fay N, Panté N. Nuclear entry of DNA viruses. Front Microbiol.2015;6:1-19.• CohenS,Au S, PanteN.Howviruses access thenucleus.BBA-MolCellBiolL.2011;1813(9):1634-45.• Krebs JE, Goldstein ES, Kilpatrick ST. Lewin's genes X. 10th ed.Sudbury,MA:JonesandBartlettPublishers;2011.• Hay RT. Adenovirus DNA replication. In: DePamphilis ML, editor.DNA replication in eukaryotic cells. Cold Spring Harbour, NY: ColdSpringHarbourLaboratoryPress;1996.p.699-719.

Replication of DNA Virus Genomes

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