Genetics & Gene Therapy Chapter 30 وسام عبد الرؤوف العايدي وسام عبد الرؤوف العايدي

The study of viral genetics : The study of viral genetics : (1) mutations and their effect on replication and patho- genesis (1) mutations and their effect on replication and patho- genesis (2) the interaction of two genetically dis- tinct viruses that infect the same cell. In addition, (2) the interaction of two genetically dis- tinct viruses that infect the same cell. In addition, (3) viruses as vectors in gene therapy and in recombi- nant vaccines, (3) viruses as vectors in gene therapy and in recombi- nant vaccines,

viral mutations Mutations in viral DNA and RNA occur by: Mutations in viral DNA and RNA occur by: (1) base substitution, (1) base substitution, (2)deletion, (2)deletion, (3) frameshift (3) frameshift

viral mutations (1)The most important practical use of mutations is in the production of vaccines containing live, attenuated virus. (1)The most important practical use of mutations is in the production of vaccines containing live, attenuated virus. These attenuated mutants have lost their patho- genicity but have retained their antigenicitymthey therefore induce immunity without causing disease.

(2) Antigenic variants such as those that occur frequently with influenza viruses, which have an altered surface protein and no longer inhibited by host preexisting antibody. The variant can thus cause disease, whereas the original strain cannot. (2) Antigenic variants such as those that occur frequently with influenza viruses, which have an altered surface protein and no longer inhibited by host preexisting antibody. The variant can thus cause disease, whereas the original strain cannot.

(3)Drug-resistant mutants, which are insensitive to an antiviral drug because the target of the drug, usu- ally a viral enzyme, has been modified.

Conditional-lethal mutations These mutations function normally under permissive condi- tions but fail to replicate or to express the mutant gene under restrictive conditions.

For example, temperature- sensitive conditional- lethal mutants express their phe- notype normally at a low (permissive) temperature, but at a higher (restrictive) temperature the mutant gene product is inactive.. For example, temperature- sensitive conditional- lethal mutants express their phe- notype normally at a low (permissive) temperature, but at a higher (restrictive) temperature the mutant gene product is inactive..

To give a specific example, tempera- ture- sensitive mutants of Rous sarcoma virus can trans- form cells to malignancy at the permissive temperature of 37°C. To give a specific example, tempera- ture- sensitive mutants of Rous sarcoma virus can trans- form cells to malignancy at the permissive temperature of 37°C. When the transformed cells are grown at the restrictive temperature of 41°C, their phenotype reverts to normal appearance and behavior. The malignant phenotype is regained when the permissive temperature is restored When the transformed cells are grown at the restrictive temperature of 41°C, their phenotype reverts to normal appearance and behavior. The malignant phenotype is regained when the permissive temperature is restored

Note that temperature-sensitive mutants have now entered clinical practice. Note that temperature-sensitive mutants have now entered clinical practice. Temperature-sensitive mutants of influenza virus are now being used to make a vac- cine, Temperature-sensitive mutants of influenza virus are now being used to make a vac- cine,

Defective interfering partides Defective interfering partides Some deletion mutants have the unusual property of being defective interfering partides. They are defective because they cannot replicate unless the deleted func- tion is supplied by a "helper" virus. They also interfere with the growth of normal virus if they infect first and preempt the required cellular functions. Defective in- terfering particles may play a role in recovery from viral infection; they interfere with the production of progeny virus, thereby limiting the spread of the virus to other cells. Some deletion mutants have the unusual property of being defective interfering partides. They are defective because they cannot replicate unless the deleted func- tion is supplied by a "helper" virus. They also interfere with the growth of normal virus if they infect first and preempt the required cellular functions. Defective in- terfering particles may play a role in recovery from viral infection; they interfere with the production of progeny virus, thereby limiting the spread of the virus to other cells.

INTERACTION (1)Recombination (1)Recombination (2)Complementation (2)Complementation (3)Phenotypic mixing (3)Phenotypic mixing

Recombination (1) Recombination is the exchange of genes between two chromosomes that is based on crossing over within regions of significant base sequence homology. (1) Recombination is the exchange of genes between two chromosomes that is based on crossing over within regions of significant base sequence homology. DNA DNA RNA RNA

Reassortment Reassortment is the term used when viruses with segmented genomes, such as influenza virus, exchange segments. This usually results in a much higher fre- quency ofgene exchange than does recombination. Reas- sortment of influenza virus RNA segments is involved in the major antigenic changes in the virus that are the basis for recurrent influenza epidemics. Reassortment is the term used when viruses with segmented genomes, such as influenza virus, exchange segments. This usually results in a much higher fre- quency ofgene exchange than does recombination. Reas- sortment of influenza virus RNA segments is involved in the major antigenic changes in the virus that are the basis for recurrent influenza epidemics.

Complementation (2) Complementation can occur when either one or both of the two viruses that infect the cell have a muta- tion that results in a nonfunctional protein (2) Complementation can occur when either one or both of the two viruses that infect the cell have a muta- tion that results in a nonfunctional protein The nonmutated virus "complements" the mutated one by making a functional protein that serves for both viruses. Complementation is an important method by which a helper virus permits replication of a defective virus The nonmutated virus "complements" the mutated one by making a functional protein that serves for both viruses. Complementation is an important method by which a helper virus permits replication of a defective virus

One clinically important example of complementation is hepatitis B virus providing its sur- face antigen to hepatitis delta virus, which is defective in its ability to produce its own outer protein. One clinically important example of complementation is hepatitis B virus providing its sur- face antigen to hepatitis delta virus, which is defective in its ability to produce its own outer protein.

This phenomenon is the basis for the complementa- tion test, which can be used to determine how many genes exist in a viral genome. This phenomenon is the basis for the complementa- tion test, which can be used to determine how many genes exist in a viral genome.

Phenotypic mixing Phenotypic mixing (3) In phenotypic mixing, the genome of virus type A can be coated with the surface proteins of virus type B (Figure 30-2). (3) In phenotypic mixing, the genome of virus type A can be coated with the surface proteins of virus type B (Figure 30-2). This phenotypically mixed virus can infect cells as determined by its type B protein coat. However, the progeny virus from this infection has a type A coat; ir is encoded solely by its type A generic material. This phenotypically mixed virus can infect cells as determined by its type B protein coat. However, the progeny virus from this infection has a type A coat; ir is encoded solely by its type A generic material.

An interesting example of phenotypic mixing is that of pseudotypes, which consist of the nucleocapsid of one virus and the envelope of another. An interesting example of phenotypic mixing is that of pseudotypes, which consist of the nucleocapsid of one virus and the envelope of another.

Pseudotype composed of the nucleocapsid of vesicular stomatitis virus (a rhabdovirus) and the envelope of human immunodeficiency virus (HIV, a retrovirus) are currently being used to study the immune response to HIV. Pseudotype composed of the nucleocapsid of vesicular stomatitis virus (a rhabdovirus) and the envelope of human immunodeficiency virus (HIV, a retrovirus) are currently being used to study the immune response to HIV.

Gene Therapy Gene Therapy Retroviruses are currently being used as vectors of the gene encoding adenine deaminase (ADA) in patients with immunodeficiencies resulting from a defective ADA gene. Retroviruses are excellent vectors because a DNA copy of their RNA genome is stably integrated into the host cell DNA and the integrated genes are ex- pressed efficiently. Retroviruses are currently being used as vectors of the gene encoding adenine deaminase (ADA) in patients with immunodeficiencies resulting from a defective ADA gene. Retroviruses are excellent vectors because a DNA copy of their RNA genome is stably integrated into the host cell DNA and the integrated genes are ex- pressed efficiently.

Recombinant Vaccines Recombinant Vaccines Recombinant viral vaccines contain viruses that have been genetically engineered to carry the genes of other viruses. Viruses with large genomes, eg, vaccinia virus, am excellent candidates for this purpose.. Recombinant viral vaccines contain viruses that have been genetically engineered to carry the genes of other viruses. Viruses with large genomes, eg, vaccinia virus, am excellent candidates for this purpose..

To construct the recombinant virus, any vaccinia virus gene that is not essential for viral replication is deleted, and the gene from the other virus that encodes the antigen that dicits neutralizing antibody is introduced. To construct the recombinant virus, any vaccinia virus gene that is not essential for viral replication is deleted, and the gene from the other virus that encodes the antigen that dicits neutralizing antibody is introduced.

For example, the gene for the surface antigen of hepatitis B virus has been introduced into vaccinia virus and is expressed in in- fected cells. Recombinant vaccines are not yet dinically available, but vaccines of this type promise to greatly improve the efficiency of our immunization programs For example, the gene for the surface antigen of hepatitis B virus has been introduced into vaccinia virus and is expressed in in- fected cells. Recombinant vaccines are not yet dinically available, but vaccines of this type promise to greatly improve the efficiency of our immunization programs

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