Lecture 3: Virus Infection and Replication PDF

Lecture 3 VIRUS INFECTION AND REPLICATION  Lecture Outline Attachment Penetration Uncoating Replication Assembly Maturation Release Virus growth curves  Virus Replication T...

Lecture 3 VIRUS INFECTION AND REPLICATION  Lecture Outline Attachment Penetration Uncoating Replication Assembly Maturation Release Virus growth curves  Virus Replication To continue the chain of infection, a virus must undergo replication in order to create new, infectious virions After gaining entry into the cell, the virion capsid breaks down, releases the genome (which is copied and used to create viral proteins), and new virus particles are assembled and released Unlike cell division, virions are assembled de novo (from scratch)  Virus Replication Cycle Seven stages in the virus replication cycle All viruses must accomplish these steps (with the exception of maturation, which only occurs in some viruses), although not necessarily in this exact order. 1. Attachment 2. Penetration 3. Uncoating 4. Replication 5. Assembly 6. Maturation 7. Release 1. Attachment  Virus Replication Cycle: Attachment Attachment: the binding of the virus to the host cell A virus first interacts with a cell at the plasma membrane Virus attachment protein attaches to a cell surface receptor Binding involves opposing electrostatic forces Some viruses require co-receptors on the cell surface (e.g., HIV) Some viruses use an initial receptor for docking and then binding to the essential receptor (e.g., HSV).  Virus Replication Cycle: Attachment The interaction: is specific for non-enveloped viruses, can occur with protruding virus attachment proteins or in Rhinovirus canyons formed by capsid (RNA) proteins for enveloped viruses, it occurs with virus attachment proteins embedded into the envelope determines the tropism of the virus  Virus Replication Cycle: Attachment Proving poliovirus uses CD155 to enter cells Mice are not normally infected with poliovirus because they do not have the poliovirus receptor Transgenic mice were created that expressed the human CD155 protein CD155-transgenic mice were able to become infected, and viral replication within the brain and spinal cord caused paralysis (as occurs with human infection) Virus Replication Cycle: Attachment and viral vector retargeting 2. Penetration  Virus Replication Cycle: Penetration Penetration: the crossing of the plasma membrane by the virus Several different methods of penetration are used by viruses  Virus entry via membrane fusion Entry by direct virus-cell membrane fusion obviously can only occur to enveloped viruses, but not all enveloped viruses enter cells by membrane fusion. Certain viruses can also transmit by cell-cell membrane fusion (cell to cell transmission). HIV is one of such examples. This has created a unique problem for vaccination, as the virus is hiding inside the cells and could not be neutralized by antiviral antibodies. Plasma membrane fusion Syncytial formation Endosome membrane fusion 3. Uncoating  Virus Replication Cycle: Uncoating Uncoating: Release of the virus genome into the cell due to the breakdown or the removal of the capsid Can occur through different mechanisms Several viruses must transport their genomes into the nucleus, and some viruses uncoat right at the nuclear envelope Nucleocapsids are transported using the microtubule system 4. Replication  Virus Replication Cycle: Replication Replication: Copying of the viral genome Replication strategy is dependent upon the type of nucleic acid genome it possesses Genomes can be DNA or RNA Genomes can be linear or circular Viral genome type: 1. dsDNA 2. ssDNA 3. dsRNA 4. +ssRNA 5. -ssRNA 6. RNA viruses that reverse transcribe Genomes can be segmented or non-segmented (more details about this in the “Influenzas virus” lecture)  Virus Replication Cycle: Replication DNA viruses All living organisms have dsDNA genomes and use DNA polymerases to copy their genomes and RNA polymerases to transcribe their genes All dsDNA viruses that infect humans enter the nucleus of the cell to use some aspect of the host machinery, e.g., Herpesviruses, adenoviruses, polyomaviruses, papillomaviruses. The exception are the poxviruses, which encode all the proteins they need for both transcription and genome replication (and so do not require entry into the nucleus) Viral DNA replication follows the same semi- consecutive replication as in cellular genome. Parvoviruses (e.g., adeno-associated virus) have a single-stranded DNA as the genome. So, the genome replicates in different way. More details on AAV lecture.  Virus Replication Cycle: RNA viruses Before we talk about RNA virus replication strategies, let’s briefly review the Central Dogma of Biology DNA is transcribed to RNA. RNA is translated to PROTEIN AAAAA  Virus Replication Cycle: RNA viruses RNA VIRUS REPLICATION BREAKS THE RULE OF THE CENTRAL DOGMA IN MANY WAYS RNA RNA RNA-dependent RNA polymerase (viral origin) RNA DNA RNA-dependent DNA polymerase - reverse transcriptase (viral origin) Then from DNA -> RNA DNA-dependent RNA polymerase (cellular) Due to these rule-breaking behaviors, all animal RNA viruses encode a polymerase, either being RNA-dependent RNA polymerase or reverse transcriptase.  Virus Replication Cycle: positive sense RNA viruses If the RNA virus has a positive sense RNA genome, then there is no issue with the initiation of gene expression as the input viral genome can be used directly for gene translation to produce viral enzymes such as RNA-dependent RNA polymerase for viral replication. Cytoplasm Proteins (including Nucleus polymerase) Translation (+ve) sense mRNA AAA Viral genome replication for virus with positive strand RNA GENOMIC (+ SENSE) RNA 5’ 3’ RNA-dependent RNA polymerase (translated from genome) (- SENSE) RNA 3’ 5’ GENOMIC (+ SENSE) RNA 5’ 3’ (For packaging into viral particles)  Virus Replication Cycle: negative sense RNA viruses If the RNA virus has a negative sense RNA genome, then the input viral genome has to be converted into plus sense before viral proteins can be produced. Thus, RNA-dependent RNA polymerase (in the protein form) must be packaged into all virions with negative-sense genome. (-ve) sense genomic RNA Polymerase (brought in by viral particle) (+ve) sense mRNA AAA Proteins RNA REPLICATION-negative strand RNA (- SENSE) RNA 3’ 5’ GENOMIC (+ SENSE) RNA 5’ 3’ (- SENSE) RNA 3’ 5’ (For packaging into viral particles)  Virus Replication Cycle: double stranded RNA viruses RNA viruses with double-stranded RNA genomes RNA polymerase must be packaged in virion, as double stranded RNA can’t serve as mRNA for direct protein translation. Proteins (+ve) sense mRNA AAA Polymerase (brought in by viral particle) Double-stranded genomic RNA RNA REPLICATION-dsRNA Double-stranded genomic RNA GENOMIC (+ SENSE) RNA 5’ 3’ (For protein translation) Double-stranded genomic RNA (For packaging into viral particles)  Virus Replication Cycle: Retroviruses RETROVIRUSES Nucleus Reverse transcriptase must be packaged in virion. dsDNA dsDNA (integration) +VE (+ve) sense mRNA RNA AAA Packaging Proteins  Summary: Replication of RNA Viruses All RNA viruses code for a polymerase, either an RNA-dependent RNA polymerase or a reverse transcriptase. If the RNA virus contains a negative-sense RNA genome, a double-stranded RNA genome, or is a retrovirus, the polymerase (in protein form) must be packaged into viral particles. This ensures that it is delivered into host cells during infection, initiating viral replication. Genome RdRp in virion? Infectivity of RNA? Initial event in cell +ssRNA No Yes Translation -ssRNA Yes No Transcription dsRNA Yes No Transcription Retrovirus Reverse Transcriptase No Reverse Transcription How do RNA viruses solve the problem of monocistronic mRNA problem, i.e., to use a single viral genome for multiple protein production? monocistronic mRNA Strategy-1: Encoding a polyprotein followed by extensive cleavage by proteinases GENOMIC (+ SENSE) RNA AAAAA translation Strategy-1: internal ribosome entry site – (IRES) Gene 1 Gene 2  Virus Replication Cycle: Replication A note on RNA viruses… RNA viruses are more prone to mutation than DNA viruses Why?? DNA-dependent DNA polymerases have proofreading ability: they can remove an incorrectly placed nucleotide and replace it with the correct one. RNA-dependent RNA polymerases do not have proofreading ability Raises the overall error rate of the enzyme, from 1 error per 109 bases for a DNA polymerase to greater than 1 error per 105 bases for an RdRp. RNA viruses have some of the highest mutation rates of all biological entities 5. Assembly 6. Maturation  HSV packaging as an example of a detailed process UL18 Scaffolding proteins (VP5, UL26/UL26.5) transported in from UL38 cytoplasm Scaffold Assembly around scaffolding proteins Mature capsid Amplified DNA Cleavage/packaging Encapsidation proteins Packaging signal: a short piece of genome region that can be recognized by the cleavage/packaging proteins.  Virus Replication Cycle: Assemble and Maturation Maturation: the final changes within an immature virion that result in an infectious virus particle Example : Within the nascent virion, the HIV Gag polyprotein becomes cleaved by the viral protease into the capsid, matrix, and nucleocapsid proteins only after the virion is released from the cell Alters the virion architecture to create an infectious virion Several HIV drugs target the HIV protease to prevent virion maturation 7. Release  Virus Replication Cycle: Release Release: the escape of nascent virions from the infected cell For enveloped viruses, release can occur through budding Assembly occurs at a membrane within the cell, with virus attachment proteins becoming embedded into the membrane Viral proteins facilitate the curving of the membrane until it becomes separated from the cell membrane, creating the viral envelope Different viruses bud from the plasma membrane, rER, Golgi complex, or vesicles  Virus Replication Cycle: Release Budding as seen under an electron microscopy Rubella virus CDC / Dr. Fred Murphy and Sylvia Whitfield  Virus Replication Cycle: Release Release can also occur via: Lysis of the cell (mostly by non- enveloped viruses  Virus Growth Curves One-step growth curves are used to study the replication cycle of a virus infection multiplicity of infection (MOI): the ratio of infectious virions to cells MOI of 1: one virus particle per cell MOI of 10: ten virus particles per cell For one-step growth curves, a high MOI is used to ensure simultaneous infection of all cells at once Eclipse period: time it takes to form infectious virions internally (these are not yet released!) Latent period: time in which no infectious viruses have been released from the cell Burst size: number of infectious virions released per cell Defective viral particles and Virus-like particles (VLPs) Defective viral particles Ratio of non-infectious to infectious particles is usually bigger than 10, indicating that the majority of the released viral particles are noninfectious (defective). Defective interfering particles, e.g., HSV amplicons (contain incomplete HSV genomic sequence), defective HIV derived from error-prone RT. Virus-like particles (VLPs) Viral particles that do not contain viral genome