Review of Medical Microbiology and Immunology, 13th Edition

30. Genetics & Gene Therapy

CHAPTER CONTENTS

Introduction

Mutations

Interactions Between Viruses

Gene Therapy & Recombinant Vaccines

Gene Therapy

Recombinant Vaccines

Pearls

Self-Assessment Questions

Practice Questions: USMLE & Course Examinations

INTRODUCTION

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

Mutations in viral DNA and RNA occur by the same processes of base substitution, deletion, and frameshift as those described for bacteria in Chapter 4. Probably the most important practical use of mutations is in the production of vaccines containing live, attenuated virus. These attenuated mutants have lost their pathogenicity but have retained their antigenicity; therefore, they induce immunity without causing disease.

There are two other kinds of mutants of interest. The first are antigenic variants such as those that occur frequently with influenza viruses, which have an altered surface protein and are therefore no longer inhibited by a person’s preexisting antibody. The variant can thus cause disease, whereas the original strain cannot. Human immunodeficiency virus and hepatitis C virus also produce many antigenic variants. These viruses have an “error-prone” polymerase that causes the mutations. The second are drug-resistant mutants, which are insensitive to an antiviral drug because the target of the drug, usually a viral enzyme, has been modified.

Conditional lethal mutations are extremely valuable in determining the function of viral genes. These mutations function normally under permissive conditions but fail to replicate or to express the mutant gene under restrictive conditions. For example, temperature-sensitive conditional lethal mutants express their phenotype normally at a low (permissive) temperature, but at a higher (restrictive) temperature, the mutant gene product is inactive. To give a specific example, temperature-sensitive mutants of Rous sarcoma virus can transform 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.

Note that temperature-sensitive mutants have now entered clinical practice. Temperature-sensitive mutants of influenza virus are now being used to make a vaccine, because this virus will grow in the cooler, upper airways where it causes few symptoms and induces antibodies, but it will not grow in the warmer, lower airways where it can cause pneumonia.

Some deletion mutants have the unusual property of being defective interfering particles. They are defective because they cannot replicate unless the deleted function 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 interfering 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.

INTERACTIONS BETWEEN VIRUSES

When two genetically distinct viruses infect a cell, three different phenomena can ensue.

(1) Recombination is the exchange of genes between two chromosomes that is based on crossing over within regions of significant base sequence homology. Recombination can be readily demonstrated for viruses with double-stranded DNA as the genetic material and has been used to determine their genetic map. However, recombination by RNA viruses occurs at a very low frequency, if at all. Reassortment is the term used when viruses with segmented genomes, such as influenza virus, exchange segments. This usually results in a much higher frequency of gene exchange than does recombination. Reassortment of influenza virus RNA segments is involved in the major antigenic changes in the virus that are the basis for recurrent influenza epidemics.

(2) Complementation can occur when either one or both of the two viruses that infect the cell have a mutation that results in a nonfunctional protein (Figure 30–1). 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 surface antigen to hepatitis delta virus, which is defective in its ability to produce its own outer protein.

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FIGURE 30–1 Complementation. If either virus A or virus B infects a cell, no virus is produced because each has a mutated gene. If both virus A and virus B infect a cell, the protein product of gene Y of virus A will complement virus B, the protein product of gene Z of virus B will complement virus A, and progeny of both virus A and virus B will be produced. Note that no recombination has occurred and that the virus A progeny will contain the mutated z gene and the virus B progeny will contain the mutant y gene. Y, Z, functional genes; y, z, mutated, nonfunctional genes.

This phenomenon is the basis for the complementation test, which can be used to determine how many genes exist in a viral genome. It is performed by determining whether mutant virus A can complement mutant virus B. If it can, the two mutations are in separate genes because they make different, complementary proteins. If it cannot, the two mutations are in the same gene, and both proteins are nonfunctional. By performing many of these paired tests with different mutants, it is possible to determine functional domains of complementation groups that correspond to genes. Appropriate controls are needed to obviate the effects of recombination.

(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; it is encoded solely by its type A genetic material. An interesting example of phenotypic mixing is that of pseudotypes, which consist of the nucleocapsid of one virus and the envelope of another. Pseudotypes 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.

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FIGURE 30–2 Phenotypic mixing. Initially, Virus 1 (Blue capsid proteins and vertical genome) and Virus 2 (Yellow capsid proteins and horizontal genome) infect the same mouse cell. Assume that Virus 1 can infect human cells but not chicken cells (a property determined by the blue surface proteins) and that Virus 2 can infect chicken cells but not human cells (a property determined by the yellow surface proteins). However, both Virus 1 and Virus 2 can infect a mouse cell. Within the mouse cell, both genomes are replicated and both blue and yellow capsid proteins are synthesized.
As shown, some of the progeny virus (Viruses 3 and 4) exhibit phenotypic mixing because they have both the blue and the yellow surface proteins and therefore can infect both chicken cells and human cells. Note that in the next round of infection, when progeny Virus 3 infects either human cells or chicken cells, the progeny of that infection (Viruses 5 and 6) is determined by the vertical genome and will be identical to Virus 1 with only blue capsid proteins and a vertical genome. Similarly (but not shown), when progeny Virus 4 infects either human cells or chicken cells, the progeny of that infection is determined by the horizontal genome and will be identical to Virus 2. (Modified and reproduced with permission from Joklik W et al. Zinsser Microbiology, 20th ed. Appleton & Lange, Norwalk, CT, 1992.)

GENE THERAPY & RECOMBINANT VACCINES

Viruses are being used as genetic vectors in two novel ways: (1) to deliver new, functional genes to patients with genetic diseases (gene therapy); and (2) to produce new viral vaccines that contain recombinant viruses carrying the genes of several different viruses, thereby inducing immunity to several diseases with one immunization.

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 expressed efficiently. Retroviral vectors are constructed by removing the genes encoding several viral proteins from the virus and replacing them with the human gene of interest (e.g., the ADA gene). Virus particles containing the human gene are produced within “helper cells” that contain the deleted viral genes and therefore can supply, by complementation, the missing viral proteins necessary for the virus to replicate. The retroviruses produced by the helper cells can infect the patient’s cells and introduce the human gene into the cells, but the viruses cannot replicate because they lack several viral genes. This inability of these viruses to replicate is an important advantage in human gene therapy.

Recombinant Vaccines

Recombinant viral vaccines contain viruses that have been genetically engineered to carry the genes of other viruses. Viruses with large genomes (e.g., vaccinia virus) are 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 elicits 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 infected cells. Recombinant vaccines are not yet clinically available, but vaccines of this type promise to greatly improve the efficiency of our immunization programs.

PEARLS

• 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.

• 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.

• Reassortment (exchange) of segments of the genome RNA of influenza virus is important in the pathogenesis of the worldwide epidemics caused by this virus.

• Complementation occurs when one virus produces a protein that can be used by another virus. A medically important example is hepatitis D virus, which uses the surface antigen of hepatitis B virus as its outer coat protein.

• Phenotypic mixing occurs when two different viruses infect the same cell and progeny viruses contain proteins of both parental viruses. This can endow the progeny viruses with the ability to infect cells of species that ordinarily parental virus could not.

SELF-ASSESSMENT QUESTIONS

1. In the lab, a virologist was studying the properties of HIV. She infected the same cell with both HIV and rabies virus. (HIV can infect only human CD4-positive cells, whereas rabies virus can infect both human cells and dog cells.) Some of the progeny virions were able to infect dog cells, within which she found HIV-specific RNA. Which one of the following is the term used to describe these results?

(A) Complementation

(B) Phenotypic mixing

(C) Reassortment

(D) Recombination

2. You have isolated two mutants of poliovirus, one mutated at gene X and the other mutated at gene Y. If you infect cells with each one alone, no virus is produced. If you infect a single cell with both mutants, which one of the following statements is most accurate?

(A) If complementation between the mutant gene products occurs, both X and Y progeny viruses will be made.

(B) If phenotypic mixing occurs, then both X and Y progeny viruses will be made.

(C) If the genome is transcribed into DNA, then both X and Y viruses will be made.

(D) Because reassortment of the genome segments occurs at high frequency, both X and Y progeny viruses will be made.

ANSWERS

1. (B)

2. (A)

PRACTICE QUESTIONS: USMLE & COURSE EXAMINATIONS

Questions on the topics discussed in this chapter can be found in the Basic Virology section of PART XIII: USMLE (National Board) Practice Questions starting on page 700. Also see PART XIV: USMLE (National Board) Practice Examination starting on page 731.