Viral Infection: Viral Life Cycle Dr. SOBIA MANZOOR Lecture 5

Viral Replication Description of Viral Replication in Two Ways: 1. Growth Curve. 2. Step-wise Events Within Cells.

Growth Curve

Virus Life Cycle Early Phase: iAttachment and entry of the virion into the host cell; iiDisassembly of the infectious particle; iiiReplication of the viral genome; Late Phase: ivReplication of virus structural components; vReassembly of the replicated pieces into progeny virus particles; viRelease from the host cell.

STEPS IN VIRAL REPLICATION 1. Attachment (adsorption) 2. Penetration 3. Uncoating 4. Genome replication 5. Assembly 6. Release

EVENT-1: Attachment/Adsorption u Antigens: reactive sites on surface, interact with specific receptors on suitable host cells. u Receptors may be proteins, carbohydrates or lipid u This is usually a passive reaction. u Host Range Specificity (some viruses has narrow range, others have broad range). u Organ Specificity u Damage to these binding sites or blocking by specific antibodies can render virions non-infectious. u Cells without the appropriate receptors are not susceptible to the virus

u The coat of enveloped viruses fuses with the host cell membrane and releases the virus nucleocapsid into the host cytoplasm. u Endocytosis (Penetration in Phenocytotic vesicle) EVENT-2:Penetration/Uptake

b. Entry via endosomes at the cell surface Enveloped Viruses a. Entry by fusing with the plasma membrane Non-enveloped Viruses may cross the PM directly or may be taken up into endosomes.

ATTACHMENT AND PENETRATION INTO CELLS The first steps in the replication process for all viruses involve the attachment of the virus particle (the virion) onto the cell surface and then the entry of at least a portion of the particle (including the genome) into the cytoplasm. Both of these steps are specific, and are the main reason for the species-specificity of viruses. For all viruses, some aspect of membrane disruption must occur to achieve entry. For enveloped viruses, this almost always involves membrane fusion between the viral envelope and a cell membrane (either the plasma membrane or an internal vesicle membrane). For non-enveloped virions, the viral surface protein(s) usually play a role in causing some very localized membrane disruption. The details of attachment and entry for many viruses are still somewhat unclear, but there are some viruses for which the process has been studied in depth.

Icosahedral virion entry: Picornavirus Poliovirus (and rhinoviruses, which cause about 50% of "colds") have an icosahedral structure which has been highly characterized. The icosahedral surface of the virion consists of 60 copies each of three proteins designated VP1, VP2, and VP3. A groove, or "canyon", in the VP1 structure provides an annulus around each pentameric vertex that provides the specific attachment site to cells. A subsequent interaction of the protein with the cell's plasma membrane leads to the entry of the viral RNA genome into the cytoplasm.

Attachment and entry of HIV Studies on HIV over the past 3 years have elucidated its entry process to the degree that it is the best understood of any human virus. The two viral envelope glycoproteins, gp120 and gp41, are responsible for attachment and membrane fusion, respectively. The cell surface glycoprotein CD4 is used as the primary receptor, and one of a few other proteins are used as co-receptors (typically CCR5 and CXCR4).

u Release of the viral genome from its protective capsid u To enable the nucleic acid to be transported within the cell u Transcription to form new progeny virions. u All purified genomes are not infectious EVENT-3:Uncoating

Gene expression u m-RNA is transcribed from viral DNA or directly from some RNA viruses and codes for viral proteins that are translated by the host cell. u 4 groups of RNA Viruses with different strategies for mRNA synthesis i. ssRNA of positive polarity (Poliovirus) ii. ssRNA of negative polarity (Measles virus, Rabies & Influenza virus) iii. dsRNA (Reovirus, 10 segments) iv. 2copies of ssRNA with positive polarity (Retroviruses) EVENT-4:Genomic activation

u Some mRNAs are translated into precursor polypeptides that must be cleaved by proteases to functional structure proteins. u Early proteins are usually non-structural (Enzymes) u Later proteins are structural. Genome Replication u Nucleic acid replication produces new viral genomes u DNA viruses replicate in nucleus (except Poxvirus) mainly with dsDNA genome (except Parvovirus). u RNA viruses mainly in the cytoplasm (except retroviruses & influenza virus). u Principle of complementarities

Viral Genome Replication Viruses utilize the flow of information of eucaryotic cells, as well as novel pathways, some which violate Central Dogma. The replication strategy of the virus depends on the nature of its genome. Viruses can be classified into seven (arbitrary) groups:

I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) Some replicate in the nucleus e.g., adenoviruses, using cellular proteins. Poxviruses replicate in the cytoplasm and make their own enzymes for nucleic acid replication. II: Single-stranded (+)sense DNA (Parvoviruses) Replication occurs in the nucleus, involving the formation of a (-) sense strand, which serves as a template for (+) strand RNA and DNA synthesis.

V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc) Have a virion associated RNA directed RNA polymerase. a) Segmented e.g. Orthomyxoviruses. The first step in replication is transcription of the (-) sense RNA genome by the virion RNA polymerase to produce monocistronic mRNAs that serve as the template for genome replication. b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced. III: Double-stranded RNA (Reoviruses; Birnaviruses) These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs. IV: Single-stranded (+) sense RNA (Picornaviruses; Togaviruses, etc) a) Polycistronic mRNA e.g. Picornaviruses; Hepatitis A. Genome RNA = mRNA. Means naked RNA is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins. b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA.

VI: Single-stranded (+) sense RNA with DNA intermediate in life-cycle (Retroviruses) Genome is (+) sense but unique among viruses in that it is diploid, and does not serve as mRNA, but as a template for reverse transcription within the newly infecting virion. VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses) Also rely on reverse transcription, but this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription.

u Packing of the viral genome within the capsid protein u No need of energy u Assembly of viral nucleocapsids may take place in the: u Nucleus (herpes virus, adenovirus) u In the cytoplasm (polio virus) u At the cell surface, "Budding" (influenza virus) EVENT-5: Assembly

u This may occur by budding from the cell surface (many enveloped viruses). u Some viruses utilize the cellular secretory pathway to exit the cell. u Virus particles enclosed within Golgi-derived vesicles are released to the outside of the cell when the transport vesicle fuses with the cell membrane. u Disintegration or lyses of the infected cell can also result in the release of intact infectious virions. EVENT-6:Release

Naked icosahedral viruses - Preassembled capsomers are joined to form empty capsids (procapsid) which are the precursors of virions. They are released from infected cells in different ways. Poliovirus is rapidly released, with death and lysis of infected cells. DNA viruses tend to mature in the nucleus tend to accumulate within infected cells over a long period and are released when the cell undergoes autolysis, and in some cases, may be extruded without lysis. MATURATION AND RELEASE Maturation proceeds differently for naked, enveloped, and complex viruses

Enveloped Viruses - Viral proteins are first associated with the nucleic acid to form the nucleocapsid, which is then surrounded by an envelope. In nucleocapsid formation, the proteins are all synthesized on cytoplasmic polysomes and are rapidly assembled into capsid components. In envelope assembly, virus-specified envelope proteins go directly to the appropriate cell membrane (the plasma membrane, the ER, the Golgi apparatus), displacing host proteins. In contrast, the carbohydrates and the lipids are produced by the host cell. The viral envelope has the lipid constitution of the membrane where its assembly takes place (eg. the plasma membrane for orthomyxoviruses and paramyxoviruses, the nuclear membrane for herpesviruses [on right]). A given virus will differ in its lipids and carbohydrates when grown in different cells, with consequent differences in physical, biological, and antigenic properties.

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