Viral Genetics and Replication SAMUEL AGUAZIM, M.D. Lange Chapter 29 & 30

Viral Growth Curve One virion infects a cell and hundreds of progeny virions are produced within hours. This is a remarkable amplification and explains the rapid spread of virus from cell to cell.

The eclipse period The eclipse period is the time when no virus particles are detected within the infected cell. It occurs soon after the cell is infected.

Cytopathic effect Cytopathic effect (CPE) is the term used to describe the damage, both morphologic and functional, inflicted on the cell by the virus. In the clinical laboratory, the presence of a virus in the patient's specimen is often detected by seeing a CPE in cell culture.

Specific events during the growth cycle

Viral Growth Cycle Attachment: The interaction of proteins on the surface of the virus with specific receptor proteins on the surface of the cell is one of the main determinants of both the species specificity and the organ specificity of the virus.

HOST-CELL RECEPTORS FOR VIRUSES Virus Receptor Polio……………......Ig superfamily protein Rabies ……………..Acetlycholine receptor Herpes……………...FGF receptor HIV………………...CD4 molecule(ccr5 &cxcr4) Influenza A…….…. Sialic acid Paramyxoviruses......Sialic acid EBV………CD 21= CR2 RHINO……ICAM1 Vaccinia…Epidermal growth factor ICAM-1 CD4 molecule Beta-adrenergic receptor

2. Fibers: projecting from the surface of adenovirus. SOME VIRAL STRUCTURES USED FOR ATTACHMENT TO CELLS 1. Surface glycoproteins: many viruses; examples-HIV, influenza, measles. 2. Fibers: projecting from the surface of adenovirus. 3. A single protein: on the surface of a capsid. 4. A mosaic: of several proteins, forming a binding-site, as in the picornaviruses.

Infectious nucleic acid is viral genome DNA or RNA, purified free of all proteins, that can undergo the entire replicative cycle within a cell and produce infectious progeny viruses. Infectious nucleic acid, because it has no associated protein, can enter and replicate within cells that the intact virion cannot.

Polarity of Viral Genome RNA: Genome RNA that has the same base sequence as the mRNA is, by definition, positive-polarity RNA. Most positive-polarity genomes are translated into viral proteins without the need for a polymerase in the virion. The exception is the retroviruses, which use reverse transcriptase in the virion to transcribe the genome RNA into DNA.

Polarity of Viral Genome RNA Genome RNA that has a base sequence complementary to mRNA has, by definition, negative polarity. A virus with a negative-polarity RNA genome must have an RNA polymerase in the virion to synthesize its mRNA.

Viral Gene Expression: All viruses require virus-specific messenger RNA to synthesize virus-specific proteins.

RNA Viruses: 1,Some RNA viruses, such as poliovirus, have a positive-polarity RNA genome that serves as the mRNA, i.e., the genome is the mRNA. 2,Other viruses, such as influenza virus, have a negative-polarity RNA genome and have an RNA polymerase in the virion that synthesizes the viral mRNA

3 , Rotavirus(Reovirus) has a double- stranded RNA genome and has an RNA polymerase in the virion that synthesizes the viral mRNA. 4,Retroviruses, such as HIV, have a positive polarity RNA genome and have a DNA polymerase in the virion that synthesizes a DNA copy of the RNA genome. This DNA is the template used by the host cell RNA polymerase to synthesize the viral mRNA.

DNA Viruses: Most DNA viruses, such as herpes viruses, adenoviruses, and papillomaviruses, have a double- stranded DNA genome and use the host cell RNA polymerase to synthesize the viral mRNA.

DNA Viruses Poxviruses have a double-stranded DNA genome but have an RNA polymerase in the virion that synthesizes the viral mRNA. Poxviruses have an RNA polymerase in the virion because they replicate in the cytoplasm and do not have access to the host cell RNA polymerase in the nucleus.

Viral Replication: All DNA viruses replicate in the nucleus, except pox viruses, which replicate in the cytoplasm. All RNA viruses replicate in the cytoplasm, except retroviruses, influenza virus, and hepatitis D virus, which require an intranuclear step in their replication. Many viruses encode a replicase, which is a DNA or RNA polymerase that synthesizes the many copies of the progeny viral genomes

Viral Genome: The genome of all DNA viruses is double-stranded except for that of parvoviruses, which is single- stranded. The genome of all RNA viruses is single-stranded except for that of reoviruses, eg, rotavirus, which is double-stranded.

Viral Proteins: Early proteins are typically enzymes used in the synthesis of viral components, whereas late proteins are typically structural proteins of the progeny viruses. Some viruses, such as poliovirus and retroviruses, translate their mRNA into precursor polyproteins, which must be cleaved by proteases to produce functional proteins.

Assembly and Release: All enveloped viruses acquire their envelope by budding through the external cell membrane as they exit the cell, except herpes viruses, which acquire their envelope by budding through the nuclear membrane. The matrix protein mediates the interaction of the nucleocapsid with the envelope.

Lysogeny Lysogeny is the process by which viral DNA becomes integrated into host cell DNA, replication stops, and no progeny virus is made. Later, if DNA is damaged by, for example, UV light, viral DNA is excised from the host cell DNA and progeny viruses are made. The integrated viral DNA is called a prophage

Lysogeny Bacterial cells carrying a prophage can acquire new traits, such as the ability to produce exotoxins such as diphtheria toxin. Transduction is the process by which viruses carry genes from one cell to another.

Lysogeny Lysogenic conversion is the term used to indicate that the cell has acquired a new trait as a result of the integrated prophage

GENETIC CHANGE MUTATION RECOMBINATION

mutation The study of viral genetics falls into two general areas: (1) mutations and their effect on replication and pathogenesis, and (2) the interaction of two genetically distinct viruses that infect the same cell. In addition, viruses serve as vectors in gene therapy and in recombinant vaccines, two areas that hold great promise for the treatment of genetic diseases and the prevention of infectious diseases.

MUTATIONS A mutation is a change in the base sequence of DNA insertion of a different amino acid into a protein and the appearance of an altered phenotype.

Mutations result from three types of molecular changes: 1. Base substitution: one base is inserted in the place of another. 2. Frame shift mutation: One or more base pairs are added or deleted. 3. When transposons or inserted sequences are integrated into DNA.

mutation Mutations in the viral genome can produce antigenic variants and drug-resistant variants. Mutations can also produce attenuated (weakened) variants that cannot cause disease but retain their antigenicity and are useful in vaccines.

mutation Temperature-sensitive mutants can replicate at a low (permissive) temperature but not at a high (restrictive)temperature. Temperature-sensitive mutants of influenza virus are used in one of the vaccines against this disease.

RECOMBINATION It is the exchange of genes between two chromosomes that is based on crossing over within regions of significant base sequence homology. It can be readily demonstrated for viruses with double stranded DNA as the genetic material and has been used to determine their genetic map.

REASSORTMENT Occurs among viruses with segmented genomes. Mixing of genome segments into new sets. Results in reassorted viruses which display properties (antigenicity, pathogenicity, etc.) differing from either parent. Especially important to epidemiology of influenza viruses.

REASSORTMENT

ORTHOMYXOVIRUSES M2 -Human HA - Human NA - Human helical nucleocapsid polymerase complex (human) lipid bilayer membrane NA - Human M2 -Human

ORTHOMYXOVIRUSES HA (bird) NA (bird) helical nucleocapsid (bird) M1 protein (bird) helical nucleocapsid (bird) HA (bird) polymerase complex lipid bilayer membrane NA (bird)

ORTHOMYXOVIRUSES HA (Human) HA (Human) HA (bird) NA (bird) M1 protein (bird) helical nucleocapsid (bird) HA (bird) polymerase complex lipid bilayer membrane NA (bird) HA (Human) HA (Human)

Non-genetic interactions: Complementation: Sharing of gene products (proteins) between two mutant viruses each virus unable to replicate alone. On infection of same cell, each produces a ‘good’ protein that is mutated in the other virus thus providing normal copies of all necessary gene products, mutant viral genome can be replicated and progeny viruses are produced.

Y z y Z

Phenotypic mixing:  a. Packaging of genome of one type of virus into capsid of another type of virus to create ‘pseudotypes’. b. Pseudotype virus has surface properties of one virus (receptor recognition and antigenicity, for example) but has the genome of a different virus.

PHENOTYPIC MIXING no changes in genome

PHENOTYPIC MIXING PSEUDOTYPE

Viral Replication Outcomes 1.Lytic infection:- virus replication in cell produces progeny virus particles, cell dies as a result. 2.Latent infection:- infected cell contains viral genome but it is not being expressed, no virus produced. The cell is not killed. Viruses can become reactivated and produce progeny virus. 3.Persistent infection: virus replicates continuously in cell and cell remains viable. 4.Transformation: virus encodes a gene product that alters the cell so that it is transformed into a cancerous or immortalized state.

Steps in viral replication.

DNA Non Enveloped Virus