VIRAL REPLICATION Lecture 2

Introduction Viral replication is the formation of biological viruses during the infection process in the target host cells. Viruses are intracellular obligate parasites which means that they cannot replicate or express their genes without the help of a living cell. A single virus particle (Virion) is in and of itself essentially inert. It lacks needed components that cells have to reproduce. When a virus infects a cell, it organizes the cell's ribosomes, enzymes and much of the cellular machinery to replicate.

Virus-Host Cell Interaction For a virus to multiply it must obviously infect a cell. Viruses usually have a restricted host range i.e., animal and cell type in which this is possible. All viruses must make proteins with 3 sets of functions Ensure replication of the genome, Package the genome into the virus particles, and Alter the metabolism of the infected cell so that viruses are produced.

Virus-Host Cell Interaction The two most commonly observed virus- host cell interactions are: The lytic interaction: which results in virus multiplication and lysis (or death) of the host cell The transforming interaction: which results in the integration (or existence as episome) of viral genome into the host genome and permanent transformation or alteration of the host cell (e.g. morphology, interaction with other cells, growth habits, etc.)

Lytic Virus-Cell Interaction During this cycle the virus enters host cell, multiplies and is released. This cycle is repeated many times when a virus particle infects an organism, until, for one reason or another, further multiplication is arrested or the host dies. The following steps is involved in a complete lytic cycle:

The receptors on cells are glycoproteins or glycolipids. Lytic Virus-Cell Interaction 1- Recognition of a target host cell (Adsorption) The receptor-binding site on virus particle reacts specifically with a corresponding receptor on a cell surface. The receptors on cells are glycoproteins or glycolipids.

Virus entry is accomplished in one of three ways: Lytic Virus-Cell Interaction 2- Internalization of the virus (Penetration and Uncoating) After virus attachment to host cell, virus penetrates the plasma membrane and releases its genome (uncoating) inside cytoplasm. Virus entry is accomplished in one of three ways: Fusion of the viral membrane and the plasma membrane with the release of viral nucleic acid into the cytoplasm (Figure 1A). Viruses have fusion proteins, e.g., measles and mumps Internalization of the whole virion by viropexis (or pinocytosis) and release of its nucleic acid (Figure 1B). Naked viruses appear to pass or slide through the external plasma membrane directly.

Lytic Virus-Cell Interaction 3- Transcription and Replication The replication strategies employed by the different viruses are different RNA viruses (+) stranded RNA acts as mRNA (-) stranded RNA, a virus associated polymerase makes a complementary (+) copies that act as mRNA DNA viruses Transcription “early” mRNA from parental DNA. Late mRNAs are transcribed from newly replicated progeny DNA molecules

Lytic Virus-Cell Interaction 4. Processing of mRNA Viral primary RNA transcripts may need to be processed before they are translated into proteins: Primary transcripts splicing is common among DNA viruses, e.g., adenoviruses. Some retrovirus and influenza virus mRNAs are also spliced. Addition of a sequence of 50-200 adenosine residues to the 3΄ terminus of the mRNA molecule. Addition of a 7-methyl guanosine “cap” to the 5΄ terminus of the transcript. Terminal signal 7-methyl guanosine “cap” : prevents degradation, transport

Lytic Virus-Cell Interaction 5. Synthesis of viral proteins Viral mRNAs are translated on normal host cell ribosomes to produce viral structural and non-structural proteins. Host cell protein synthesis machinery is responsible for reading the genetic message of the viral mRNAs, in a triplet code with start and stop codons, as in reading normal cellular mRNAs.

Lytic Virus-Cell Interaction 5. Synthesis of viral proteins A single viral protein is synthesized from a single mRNA, Some viruses have a second strategy, whereby a very large viral poly-protein is first formed, which is then cleaved at specific sites by viral or cellular proteolytic enzymes to give a series of smaller viral proteins. In a third strategy, two virus proteins may be encoded by a single mRNA (see overlapping genes) since the mRNA may be read in different reading frames.

Lytic Virus-Cell Interaction 6. Assembly Virus structural proteins, such as envelope and matrix proteins, migrate to the plasma membrane (budding viruses) or, alternatively, may assemble in the cell cytoplasm (lytic viruses) or the nucleus. Carbohydrates and other groups are added to newly synthesized proteins by cellular enzymes.

Lytic Virus-Cell Interaction 7. Release In case of budding, viral proteins are inserted in the external plasma membrane of the host cell. The proteins and nucleic acid assemble in the host cell and bud by protrusion through the cellular plasma membrane The cell may continue to produce successive waves of new viruses, as many as 10,000 virions may be produced per cell in as a few as 6 hours. Some viruses, e.g., poliovirus, may assemble completely in the cytoplasm and are released only after lysis and death of the cell.

GENETIC VARIATION OF VIRUSES

Two principal mechanisms are involved: Since viruses are made up of nucleic acid molecules surrounded by a protective coat, they undergo mutations, interact with the host nucleic acid, and interact with other viruses in mixed infected cells. This chapter covers the mechanisms by which genetic changes occur in viruses. Two principal mechanisms are involved: Mutation and recombination

Mutations In general, the base sequence of a genome must be preserved from one generation to the next, otherwise progeny might not be able to synthesize all the proteins required for their own survival and reproduction. Yet changes do occur in the genomes, and they may affect one or more characters of the organism. Said another way, they give rise to variation in phenotype.

Mutations One mechanism by which this change occurs is called gene mutation. This is a stable change in the nucleotide sequence of the organism genome. This might be a deletion, addition, or substitution of one to several bases in the sequence of a gene. Even a change in a single nucleotide might lead to significant consequences!

Mutations Induced Mutations Spontaneous Mutations Some gene mutations are induced by mutagens, environmental agents that can attack a nucleic acid molecule and modify its structure. Ultraviolet, radiation, ionizing radiation and certain chemicals are examples of mutagens. Spontaneous Mutations Other gene mutations are spontaneous; they are not induced by agents outside the cell or organism. These are the mutations which occur naturally during viral replication.

Mutations Virus Mutants “Strain”, “type”, “mutant” and even “isolate” are all terms used interchangeably to differentiate them from original “parental”, “wild type” or “street” viruses.

Mutations Mutation Rates and Outcomes DNA viruses have spontaneous mutation rates similar to those of eukaryotic cells because, like eukaryotic DNA polymerases, their replication enzymes have proofreading functions. The error rate for DNA viruses is about 10-8 to 10-11 error per incorporated nucleotide. With this low mutation rate, replication of even the most complex DNA viruses, which have 2x105 to 3x105 nucleotide pairs per genome, will generate mutants rather rarely.

Mutations The RNA viruses, however, lack a proofreading function in their replication enzymes, and some have mutation rates of 10-3 to 10-4 per incorporated nucleotide. Even the simplest RNA viruses, which have about 7400 nucleotides per genome, will generate mutants frequently Not all mutations that occur persist in the virus population. Mutations that interfere with the essential functions of multiplication cycle are rapidly lost from the population. Only mutations that do not cripple essential viral functions can persist or become fixed in a virus population

Phenotypic Variation by Mutations Mutations that alter the viral phenotype but are not deleterious may be important. For example mutations can create novel antigenic determinants which may then enable the virus to infect a previously immune host e.g H5N1, H1N1, H3N1 strains of influenza virus. Additionally, mutation has been a principal tool in developing attenuated live virus vaccines