Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 60
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Plant viruses can be assayed similarly by counting local lesions on plant leaves inoculated with viruses.Analysis of viral mutants, which are commonly isolated byplaque assays, has contributed extensively to current understanding of molecular cellular processes. The plaque assayalso is critical in isolating bacteriophage clones carryingsegments of cellular DNA, as discussed in Chapter 9.Lytic Viral Growth Cycles Lead to Deathof Host CellsAlthough details vary among different types of viruses, thosethat exhibit a lytic cycle of growth proceed through thefollowing general stages:1. Adsorption—Virion interacts with a host cell by bindingof multiple copies of capsid protein to specific receptors onthe cell surface.2. Penetration—Viral genome crosses the plasma membrane.For animal and plant viruses, viral proteins also enter thehost cell.3.
Replication—Viral mRNAs are produced with the aidof the host-cell transcription machinery (DNA viruses) orby viral enzymes (RNA viruses). For both types of viruses,viral mRNAs are translated by the host-cell translationmachinery. Production of multiple copies of the viralgenome is carried out either by viral proteins alone orwith the help of host-cell proteins.4. Assembly—Viral proteins and replicated genomesassociate to form progeny virions.5. Release—Infected cell either ruptures suddenly (lysis),releasing all the newly formed virions at once, or disintegrates gradually, with slow release of virions.Figure 4-40 illustrates the lytic cycle for T4 bacteriophage, a nonenveloped DNA virus that infects E. coli. Viralcapsid proteins generally are made in large amounts becausemany copies of them are required for the assembly of eachprogeny virion. In each infected cell, about 100–200 T4progeny virions are produced and released by lysis.The lytic cycle is somewhat more complicated for DNAviruses that infect eukaryotic cells.
In most such viruses, theDNA genome is transported (with some associated proteins)into the cell nucleus. Once inside the nucleus, the viral DNAis transcribed into RNA by the host’s transcription machinery. Processing of the viral RNA primary transcript by hostcell enzymes yields viral mRNA, which is transported to thecytoplasm and translated into viral proteins by host-cellribosomes, tRNA, and translation factors. The viral proteinsare then transported back into the nucleus, where some ofthem either replicate the viral DNA directly or direct cellular proteins to replicate the viral DNA, as in the case of SV40discussed in the last section.
Assembly of the capsid proteinswith the newly replicated viral DNA occurs in the nucleus,yielding hundreds to thousands of progeny virions.Most plant and animal viruses with an RNA genome donot require nuclear functions for lytic replication. In some140CHAPTER 4 • Basic Molecular Genetic Mechanisms FIGURE 4-40 Lytic replicationLysis/releasecycle of E.
coli bacteriophage T4, anonenveloped virus with a doublestranded DNA genome. After viral coatproteins at the tip of the tail in T4interact with specific receptor proteinson the exterior of the host cell, the viralgenome is injected into the host (step 1 ).Host-cell enzymes then transcribeviral “early” genes into mRNAs andsubsequently translate these into viral“early” proteins (step 2 ). The earlyproteins replicate the viral DNA andinduce expression of viral “late” proteinsby host-cell enzymes (step 3 ). The virallate proteins include capsid and assembly proteins and enzymes that degradethe host-cell DNA, supplying nucleotidesfor synthesis of more viral DNA. Progenyvirions are assembled in the cell (step 4 )and released (step 5 ) when viral proteinslyse the cell.
Newly liberated virusesinitiate another cycle of infection in otherhost cells.Adsorption/injection5E. colichromosome1Free virionT4 DNAExpression2 of viral earlyproteinsAssembly 4Viralproteins3Replication of viral DNAExpression of viral late proteinsRabies virusNucleocapsid proteinLipid bilayerMatrix proteinGenomic RNAReceptor-binding glycoproteinViral RNA polymerase1 AdsorptionBudding 13Virus receptorFusion 912Associationat membraneCytosol2 Endocytosis11GolgiProgeny capsidassemblyEndosomeTransport 8Matrix andnucleocapsid 10synthesis3 FusionViralmRNA7GlycoproteinsynthesisERNucleus5 Replication6TranscriptionCell membrane4Release4.7 • Viruses: Parasites of the Cellular Genetic Systemof these viruses, a virus-encoded enzyme that enters the hostduring penetration transcribes the genomic RNA intomRNAs in the cell cytoplasm.
The mRNA is directly translated into viral proteins by the host-cell translation machinery. One or more of these proteins then produces additionalcopies of the viral RNA genome. Finally, progeny genomesare assembled with newly synthesized capsid proteins intoprogeny virions in the cytoplasm.After the synthesis of hundreds to thousands of new virions has been completed, most infected bacterial cells andsome infected plant and animal cells are lysed, releasing allthe virions at once.
In many plant and animal viral infections, however, no discrete lytic event occurs; rather, the deadhost cell releases the virions as it gradually disintegrates.As noted previously, enveloped animal viruses are surrounded by an outer phospholipid layer derived from theplasma membrane of host cells and containing abundantviral glycoproteins. The processes of adsorption and releaseof enveloped viruses differ substantially from these processesin nonenveloped viruses. To illustrate lytic replication ofenveloped viruses, we consider the rabies virus, whose nucleocapsid consists of a single-stranded RNA genome surrounded by multiple copies of nucleocapsid protein.
Like FIGURE 4-41 Lytic replication cycle of rabies virus, anenveloped virus with a single-stranded RNA genome. Thestructural components of this virus are depicted at the top. Notethat the nucleocapsid is helical rather than icosahedral. After avirion adsorbs to multiple copies of a specific host membraneprotein (step 1 ), the cell engulfs it in an endosome (step 2 ).A cellular protein in the endosome membrane pumps H ionsfrom the cytosol into the endosome interior.
The resultingdecrease in endosomal pH induces a conformational change inthe viral glycoprotein, leading to fusion of the viral envelopewith the endosomal lipid bilayer membrane and release of thenucleocapsid into the cytosol (steps 3 and 4 ). Viral RNApolymerase uses ribonucleoside triphosphates in the cytosol toreplicate the viral RNA genome (step 5 ) and to synthesize viralmRNAs (step 6 ). One of the viral mRNAs encodes the viraltransmembrane glycoprotein, which is inserted into themembrane of the endoplasmic reticulum (ER) as it is synthesizedon ER-bound ribosomes (step 7 ). Carbohydrate is added to thelarge folded domain inside the ER lumen and is modified as themembrane and the associated glycoprotein pass through theGolgi apparatus (step 8 ).
Vesicles with mature glycoprotein fusewith the host plasma membrane, depositing viral glycoproteinon the cell surface with the large receptor-binding domain outside the cell (step 9 ). Meanwhile, other viral mRNAs are translated on host-cell ribosomes into nucleocapsid protein, matrixprotein, and viral RNA polymerase (step 10 ). These proteins areassembled with replicated viral genomic RNA (bright red) intoprogeny nucleocapsids (step 11), which then associate with thecytosolic domain of viral transmembrane glycoproteins in theplasma membrane (step 12). The plasma membrane is foldedaround the nucleocapsid, forming a “bud” that eventually isreleased (step 13).141▲ EXPERIMENTAL FIGURE 4-42 Progeny virions ofenveloped viruses are released by budding from infectedcells.
In this transmission electron micrograph of a cell infectedwith measles virus, virion buds are clearly visible protruding fromthe cell surface. Measles virus is an enveloped RNA virus with ahelical nucleocapsid, like rabies virus, and replicates as illustratedin Figure 4-41. [From A. Levine, 1991, Viruses, Scientific AmericanLibrary, p. 22.]other lytic RNA viruses, rabies virions are replicated in thecytoplasm and do not require host-cell nuclear enzymes. Asshown in Figure 4-41, a rabies virion is adsorbed by endocytosis, and release of progeny virions occurs by buddingfrom the host-cell plasma membrane.
Budding virionsare clearly visible in electron micrographs of infected cells, asillustrated in Figure 4-42. Many tens of thousands of progeny virions bud from an infected host cell before it dies.Viral DNA Is Integrated into the Host-CellGenome in Some Nonlytic Viral Growth CyclesSome bacterial viruses, called temperate phages, can establisha nonlytic association with their host cells that does not killthe cell.
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