Chapter 4

Viral attachment protein

(or receptor-binding protein) is the virus ligand that binds to the virus receptor Located on the outer surface of virion

Size and composition of virus genome can dictate:

1. Degree of dependence on host cell for replication 2. Strategies of gene expression 3. Interactions with host Viral pathogenesis

Temporal regulation in herpesviruses- late genes

Expressed after RNA replication Down regulate early genes Virus structural proteins Orchestrate assembly and release of virions

Penetration by Non-enveloped viruses

No envelope= no fusion Penetrate at plasma membrane or thru endosome

RNA virus genes are expressed at similar levels throughout the replication cycle

No temporal difference in gene expression Exception is some retroviruses: replicate through DNA intermediate. Temporal pattern of early and late genes. Regulated post-transcriptionally

Lysis

The virus particles induce dissolution of the endosomal membrane Virus capsid/genome released into cytosol eg. Adenovirus

polarity of rNA viruses

by definition, mRNA is positive sense and negative is complementary to mRNA

virus polarity

by definition, mRNA is positive sense and negative is complementary to mRNA; Positive, negative, or ambisense

Biosynthesis of viral nucleic acid and proteins: pathogenesis

insights into in vivo "life-style"

Release from cell-Enveloped viruses

may or may not require cell lysis Assemble adjacent to cell membrane and mature by budding Process of budding is not necessarily lethal to cell, though it often is. Non-infectious until the virion acquires an envelope.

Penetration across a cellular membrane

membrane fusion (only enveloped viruses) Membrane penetration.

Nature of viral RNA genomes

strandedness, segmented, polarity

Primary receptors

Binding results in changes in virus proteins and/or the plasma membrane that lead to entry of the virus into the cell

Release from cell: non-enveloped viruses

Cell lysis: the cell dies, membrane degrades, and virions are released

Virus: host interactions

Change in receptor usage can result in expansion of host range and newly emerging disease

Virus receptor/ host cell receptor

can be defined as cell-surface molecule that binds the incoming virus to the cell and promotes entry Defines host cell range

Pathways for mRNA synthesis by DNA viruses

dsDNA viruses: use cellular RNA polymerase 2 or virion transcriptase to make mRNA. ssDNA viruses: Cellular DNA polymerase>> double stranded DNA>> Cellular RNA polymerase 2

Viral cell interactions: papillomavirus

dsDNA, circular Non-enveloped No virion DNA polymerase 7-8 kb, requires cell to be in S phase for replication Infects basal epithelial keratinocytes, INDUCES entrance into S phase of cell cycle. Viral proteins inactivate tumor suppressor genes. Clinical outcome: warts, cervical cancer

Advantages of temporal regulation

most often seen in large DNA viruses Competition with host cell for gene expression Delay expression of highly antigenic structural proteins

Envelope glycoproteins

signal peptide on N-terminus Targets to the ER and golgi, Modified by addition of carbohydrates Cleaved by cellular proteases

Perforation

The entire capsid is passed through membrane No major lysis of the membrane eg. Parvovirus, Rotavirus

Viral cell interactions: canine parvovirus

ss DNA, linear Non-enveloped, no virion DNA polymerase, 5 kb, requires cells to be in S phase for replication. Replicates in rapidly dividing cells that are often in S phase Clinical outcome: leukopenia, hemorrhagic diarrhea

Pathways for mRNA synthesis by RNA viruses

ssRNA viruses, positive sense: viral genomes act as mRNA dsRNA: use virion RNA-dependent RNA polymerase. ssRNA viruses, negative sense: use virion RNA-dependent RNA polymerase. diploid RNA: virion reverse transcriptase>> dsDNA>> cellular RNA polymerase 2>> mRNA Ambisense ssRNA: use virion RNA-dependent RNA polymerase.

Virus: cell interactions

The presence or absence of activating proteases can be important determinant of tropism and virulence. Specificity of proteolytic cleavage site is an important virulence determinant

Process to determine One step growth curve

1. Inoculate cultured mammalian cells with virus at a high multiplicity of infection (MOI) 2. At varying times post-inoculation, sample cells/culture supernatant 3. Determine amount of virus produced by titration on new fresh cells 4. Plot amount of virus over time

EHV-1; Single amino acid change in viral DNA polymerase

10-fold increase in virus replication More severe morbidity and mortality increased shedding and more efficient spread No difference in antigenic composition, but not controlled by vaccines

Unique features of DNA virus replication: Assembly and release

DNA virus assembly in nucleus. Nothing particularly unique about DNA virus release that differs from RNA viruses.

Regulation of Gene expression by DNA viruses

DNA viruses typically display temporal regulation of virus gene expression. Different classes of viral genes (and proteins) are synthesized at sequential times. Highly regulated and interdependent 'waves' or cascades of proteins. Ordered in expression. Later genes downregulate early genes

pathogenesis of viral disease

Diseases caused by viruses usually result from direct effect of virus replication in its host cell Understanding virus-cell interactions during steps of virus replication is key to understanding pathogenesis of viral diseases

Equine Herpesvirus neurological disease

EHV-1 associated with respiratory, reproductive, and neurological disease. Can cause abortion, neurologic signs. Recent outbreaks of highly neurovirulent form of EHV-1 at racetracks, show venues, clinics, and boarding stables across the country

viral RNA polymerases

Either packaged into the virion the transported into the newly-infected cell, or synthesized very soon after infection. Lack proofreading and have a high error rate during genome replication. Excellent targets for antiviral drugs

Steps of the virus replication cycle in each phase

Entry phase: Attachment and penetration Eclipse phase: uncoating, biosynthesis Release phase: assembly and release

fusion proteins- cleavage by a cellular protease

Envelope glycoprotein is often translated as a precursor polyprotein Cleavage of the polyprotein by cellular proteases: N-terminus location of fusion peptide. Rearrangement of the protein Additional conformation changes usually required for full activation

membrane fusion

Enveloped viruses can penetrate via membrane fusion. Envelope is derived from cellular membrane. Fusion between viral and cell membrane allows entry into cytoplasm. Depending on virus, fusion occurs at either; plasma membrane, endosomal membrane. Fusion is initiated by virus-encoded fusion proteins.

Temporal regulation in herpesviruses- immediate early genes

Expressed at start of infection Encode regulatory proteins/transcription factors; activate expression of early and late viral genes, can activate expression of certain cellular genes. "set the table" for replication of viral genome

Temporal regulation in herpesviruses- early genes

Expressed prior to DNA replication Encode enzymes, polymerases, etc. required for DNA replication Encode proteins that modulate host cell environment

Change in receptor binding and expanded host range

Feline panleukopenia and canine parvovirus Gained ability to bind to canine transferrin receptor and causes myocarditis and enteritis Canine virus 2a infect both cats and dogs

Significance of the Eclipse Phase

Illustrates a fundamental feature of viruses It's presence tells us that this is a virus

Clinically-relevant aspects of viral fusion proteins

Important determinants of tissue tropism, host range and virulence Important targets for host immune defenses, such as neutralizing antibodies, cytotoxic T-cells Important targets for antiviral drugs Lead to 'syncytial cell' formation by fusing cells

RNA viruses must compete with cell genes for translation of viral mRNA

Interference with formation of the 5'- cap on the cellular mRNA. Removal of the 5'-cap from the cellular mRNA Utilize internal ribosome entry sites (IRES), eg. BVDV

Unique features of DNA virus replication: Biosynthesis of viral nucleic acid and proteins

Larger genomes- greater coding capacity- particularly herpes, pox, adenovirus. Temporal patterns of gene regulation are characteristic of larger DNA viruses. Productive infections by DNA viruses are always cytocidal. Most DNA viruses are potentially oncogenic (exception parvoviruses). Replication usually occurs in nucleus (exception poxvirus)

Co-receptors

May differ from the primary receptor in: binding affinity. Cell/tissue distribution. Can aid immune evasion

trypsin like proteases

Non-pathogenic viruses have a single arginine residue at the HA0 cleavage site cleaved by these proteases that are present in a limited number of cell or tissue types. Replication of low-pathogenic influenza virus is limited to these sites, and so these viruses normally cause localized infections.

Receptor-mediated endocytosis

Normal cellular process for uptake of macromolecules via specific receptors; recycle receptors to plasma membrane degrade proteins through lysosomal pathway- series of vesicles with decreasing pH. Acidification of vacuole Hijacked by viruses to gain entry and initiation infection Pathway used by both enveloped and non-enveloped viruses. Advantages of receptor-mediated endocytosis: Provides environment (low pH) that promotes fusion or uncoating. Facilitate intracellular trafficking to perinuclear region, a "free ride through cytoskeleton"

Unique features of DNA virus replication: Attachment, penetration/uncoating

Nothing particularly unique about DNA viruses that differs from RNA viruses.

Unique features of RNA virus replication: Attachment, penetration/uncoating

Nothing particularly unique about RNA viruses that differs from DNA viruses

Virus-receptor interaction: a point of virus vulnerability

One of the best ways to defend against viruses is to prevent virus infection by blocking entry into cells Antibody that blocks binding of the virus to it's receptor can neutralize virus infectivity Some viruses have evolved a strategy wherein the binding site for receptor molecules are "masked", or located where antibody cannot reach

Furin-like proteases

Pathogenic viruses have multiple arginines/basic residues at the HA0 cleavage site, which is cleaved by these proteases. These are ubiquitously expressed in a variety of different cell types. This allows for the systemic spread of the virus in infected hosts and increases the likelihood of serious disease.

Genome replication in RNA viruses

Production of progeny RNA virus genomes requires virus encoded RNA-dependent polymerases Most RNA viruses use RNA-dependent RNA polymerase (RdRp); synthesis of complementary RNA, which serves as a template for making more viral RNA. When viral RNA is negative sense, the complementary RNA will be positive sense. When viral RNA is positive sense, the complementary RNA will be negative sense Retroviral RNA polymerase (reverse transcriptase, RdDp) synthesizes DNA, which serves as template for RNA polymerase 2 synthesis of viral genomic RNA.

Diversity of virus genome forms/structures

RNA or DNA Linear or circular (circovirus) Single or multiple segments. Strandedness: double-stranded (ds) or single-stranded (ss)

Unique features of RNA virus replication: Assembly and release

RNA virus assembly is usually in cytoplasm, Nothing particularly unique about RNA virus release that differs from DNA viruses.

What are the only organisms that use RNA as their genetic material?

RNA viruses are only organisms known to use RNA as their genetic material

Functional significance of processing of viral mRNAs and proteins

Receptor binding-site Antigenic site- masking Fusion peptide primed for activation Targets protein to assembly location

Release

Release of virus particles, or virions, from the cell May be consequence of cell lysis Some enveloped viruses may bud from the cell (does not necessarily kill the cell) Not all released viral particles are infectious. The ratio of non-infectious to infectious particles varies with the virus and the growth conditions

Attachment factors

Serve to bring virus into direct contact with the cell surface Usually do not actively promote entry

Assembly location

Sites of replication and assembly may differ, but. RNA viruses: cytoplasm. DNA viruses: nucleus. Site of assembly often site where inclusion bodies are seen in the cell.

Unique features of RNA virus replication: biosynthesis of viral nucleic acid and proteins

Synthesis of viral mRNA and replication of viral genomes requires viral coded enzymes-packaged or synthesized early in infection. RNA polymerases are more error prone, resulting in a higher mutation rate than DNA viruses- Large genomes are rare- exception Coronaviruses. Regulation of gene expression in RNA viruses is relatively minor (exception some retroviruses), and occurs post-transcriptionally Genomes of retroviruses are transcribed into DNA by reverse transcriptase. RNA viral genomes may be segmented or continuous. Segmented genomes occur only in RNA viruses. Replication usually occurs in the cytoplasm.

Genome replication of DNA viruses

Synthesis of virus DNA genomes same basic mechanism as replication of cellular DNA "Variation on a theme" among different virus families Strategies for initiating replication/ replicating termini; complementary termini (repeats/hairpins), protein primers attached to 5'-termini, circular DNA genome

importance of viral receptors

The distribution and expression of virus receptors also define, in part, the pathogenic potential of a virus as well as the nature of ensuing disease

The canyon hypothesis

The receptor-binding site is located at the bottom of a "canyon" on the viral capsomer. An an antibody molecule is too big to enter the canyon; therefore, the receptor binding site is "protected" from neutralizing antibody. The cellular receptor is long and slim, and therefore can access viral receptor binding site. Neutralizing antibody can bind the "rim" of the canyon and block receptor binding. Antigenic variation on canyon rim does not alter receptor-binding specificity, which is determined by amino acids on the "canyon floor"

Uncoating

The viral nucleic acid is released from the capsid to begin virus replication. Can be inhibited by antibody or drugs. When the nucleic acid is uncoated, infectious virus particles cannot be recovered from the cell- this is the start of the Eclipse phase. Can occur at the same time as penetration. One of the least understood steps in virus replication. Occurs usually in the cytoplasm, sometimes in the vacuole or nucleus. Aided by viral or cellular enzymes, or a drop in pH.

Virion Assembly

The virus proteins and genomic nucleic acid must co-localize and assemble into a virus particle. 1. Formation of individual structural units of protein shell; Capsomer- Self assembles 2. Assembly of the protein shell through interactions among structural units. 3. Selective packaging of nucleic acid genome and other essential viral components. 4. Acquisition of an envelope (for enveloped viruses)

Synthesis of viral proteins and nucleic acid

Transcription of viral mRNA Replication of viral nucleic acid Translation of viral protein many strategies employed Proteins for for replication and components of the virion structure. New genomes for packaging into virions Synthesize mRNAs recognized by cell translation machinery Regardless of form of nucleic acid, each need to get to mRNA recognized by host translation machinery. Strategies of virus replication organized into a framework centered around viral mRNA

Activation of fusion proteins

Triggering events for activation of fusion protein 1. Cleavage by a cellular protease. 2. Low pH-induced conformational changes (RME) 2. Receptor-binding-induced conformational changes

Replication and expression of RNA viruses

Use virus-encoded enzymes for replication of RNA by one of two unique biochemical pathways. RNA-dependent-RNA synthesis (RNA replication) RNA-dependent-DNA synthesis (reverse transcription- used by retroviruses, reverse transcriptase). Neither pathway is normally found in host cells: enzymes used in these pathways must be encoded by the virus. May be brought in virion, or expressed during infection. Excellent targets for antiviral drugs

Assembly/ maturation

Viral nucleic acid and viral proteins are assembled to form a new virion Some viruses require a maturation step that follows the initial assembly process.

Targeting to Assembly Sites

Viral proteins and nucleic acid contain signals that aid in trafficking and packaging. Interact with cellular proteins to traffic virus component to appropriate cellular location; Vesicle trafficking, Microtubules Signals facilitate interactions among viral compounds to ensure proper components are assembled and packaged

fusion proteins

Viral-encoded proteins that induce fusion between viral envelope and cellular membrane. Envelope glycoproteins that contain hydrophobic domains, sometimes called fusion peptides, which insert into the cell membrane. In native conformation of the envelop glycoprotein, the fusion peptide is in an inactive conformation. Activated by conformational changes that free the fusion peptide for insertion into cellular membrane. Conformational changes and activation of the fusion protein occur by a variety of mechanisms.

Attachment

Virus particle attaches to the cell surface The viral attachment protein recognizes specific receptors on the outside of the cell (protein, carbohydrate or lipid). Cell receptors may be protein carbohydrate or lipid. Attachment occurs via ionic interaction. Attachment is temperature-independent. Cells without the appropriate receptors are not susceptible to the virus infection Virus replication begins with binding and attachment of virion to host cell. Mediated by ionic interactions between virus attachment proteins and virus receptors on host cell. A primary, but not the only, determinant of host cell specificity... and therefore of tissue specificity and species specificity Viruses may bind multiple molecules on cell surface.

Membrane puncture

Virus particle generates a pore in membrane Genome, but not capsid, it is released into cytosol eg. Picornaviruses

Penetration

Viruses enter the cell in a variety of ways according to the nature of the virus Virus penetrates the cell by passing through a cellular membrane; At the cell surface (plasma membrane). From within a vacuole (endosome) Penetration occurs in a variety of ways including: Membrane fusion (enveloped viruses). Membrane permeabilization (non-enveloped viruses). These are temperature dependent. The entire viron, a portion of the virion, or only the nucleic acid may penetrate into the cell. Penetration and uncoating may occur simultaneously.

Strategies to maximize viral coding capacity

Viruses genomes violate the "one gene, one mRNA" dogma that dominates in mammalian cells. poly proteins polycistronic mRNAs- polyproteins, overlapping reading frames, splicing/alternative splicing of mRNA, alternate translational start sites, translational frameshifting. Viral proteases that cleave polyproteins are a target for antiviral drugs.

Biosynthesis of viral nucleic acid and proteins: diversity

viral genomes Strategies of replication- common features based on nature of nucleic acid. Within families there are common strategies, with variations among different species.

Biosynthesis of viral nucleic acid and proteins: vulnerability

viral polymerases/ proteases are targets for antiviral drugs