Ribavirin and Influenza Case File

Ribavirin and Influenza Case File

Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

❖ CASE 2

A 21-year-old college student presents to the clinic complaining of a sudden onset of chills and fever, muscle aches, headache, fatigue, sore throat, and painful nonproductive cough 3 days prior to fall final exams. Numerous friends of the patient in the dormitory reported similar symptoms and were given the diagnosis of influenza. He said that some of them were given a prescription for ribavirin. On examination, he appears ill with temperature 39.4°C (103°F). His skin is warm to the touch, but no rashes are appreciated. The patient has mild cervical lymph node enlargement but otherwise has a normal examination.

◆ What is the most likely diagnosis?

◆ What is the biochemical mechanism of action of ribavirin?

◆ What is the genetic make up of this infectious organism?

ANSWERS TO CASE 2: RIBAVIRIN AND INFLUENZA

Summary: A college student complains of the sudden onset of fever, chills, malaise, nonproductive cough, and numerous sick contacts in the fall season.

◆ Likely diagnosis: Acute influenza infection

◆ Biochemical mechanism of action of ribavirin: A nucleoside analogue with activity against a variety of viral infections

◆ Genetic makeup of organism: Ribonucleic acid (RNA) respiratory virus

CLINICAL CORRELATION

This 21-year-old college student has the clinical clues suggestive of acute influenza. Typically, the illness occurs in the winter months with an acute onset of fever, myalgias (muscle aches), headache, cough, and sore throat. Usually, there are outbreaks with many individuals with the same symptoms. This patient is young and healthy, and antiviral therapy is not mandatory. The best way to prevent the infection is by influenza vaccination, usually given in October or November of each year. Because of the antigenic changes of the virus, a new vaccine must be given each year. Patients who are at especially high risk for severe complications or death should receive the vaccine each year. These include the elderly and people with asthma, chronic lung disease, human immunodeficiency virus (HIV) infection, diabetes, or chronic renal insufficiency.

APPROACH TO THE USE OF RIBAVIRIN IN INFLUENZA

Objectives

1. Know the structure of deoxyribonucleic acid (DNA) and RNA.

2. Understand the processes of denaturation, renaturation, and hybridization of DNA.

3. Know the differences between RNA and DNA.

4. Be familiar with the differences between human and viral/bacterial DNA and RNA.

Definitions

Base pairing: The hydrogen bonds formed between complementary bases that are part of the polynucleotide chains of nucleic acids. The base pairing is specific in that adenine will base pair with thymine (uracil in RNA) and guanine will pair with cytosine.

Chargaff rule: the amount of adenine and thymine in DNA is equal; the amount of cytosine and guanine are equal. (A = T, C = G). The amount of purines equals the amount of pyrimidines.

Helicase: An enzyme that will catalyze the separation of the strands of the DNA double helix during replication.

Nucleoside: A nitrogenous base (purines such as adenine or guanine; pyrimidines such as uracil, thymine, or cytosine) in an N-β-glycosidic linkage to a pentose sugar (deoxyribose in DNA and ribose in RNA).

Nucleotide: A nucleoside that has a phosphoester bond to one of the hydroxyl groups of the pentose sugar.

Polymerase: An enzyme that will add nucleotides to a growing nucleic acid chain using a template strand to determine which nucleotide is added. A nucleoside triphosphate condenses with the growing strand releasing pyrophosphate.

Ribavirin: 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide; a purine nucleoside analog that exhibits antiviral activity against a broad spectrum of DNA and RNA viruses.

DISCUSSION

DNA and RNA are both polymers of nucleosides joined by 3',5'-phosphodiester linkages. Each nucleoside consists of a heterocyclic nitrogenous base in a glycosidic link with a pentose sugar. The backbone of both DNA and RNA is formed by the phosphate bridges between the 3'-hydroxyl group of one pentose and the 5'-hydroxyl group of another. The nitrogenous bases form the “side chains.” DNA contains the bases adenine (A), guanine (G), cytosine (C), and thymine (T), whereas RNA contains A, G, and C but has uracil (U) instead of T. The pentose sugar in DNA is 2'-deoxyribose, whereas in RNA it is ribose.

The most stable DNA structure is formed when two polynucleotide chains are joined by hydrogen bonding between the side chain bases. The base pairing is specific in that adenine forms hydrogen bonds with thymine, whereas guanine forms hydrogen bonds with cytosine. The result is an antiparallel double helix in which one polynucleotide strand runs in the 5' to 3' direction, while the other runs in the 3' to 5' direction. The phosphate groups are located on the outside of the double helix with the base pairs forming the “stair steps” in the center of the spiral. RNA, on the other hand, is usually single stranded (the exception is certain RNA viruses), but the strand can loop back on itself and form regions of base pairing (A with U and G with C). The presence of the hydroxyl group at the 2'-position makes RNA much more susceptible to hydrolysis and decreases its stability.

The double helical structure of DNA must be disrupted during almost all biological processes in which it participates, including DNA replication and repair, as well as transcription of the DNA sequence information to RNA. Experimentally, the double helix can be separated, or denatured, by increasing the temperature to well above 50°C (122°F). If the temperature is carefully decreased, renaturation occurs when the base pairs reform. Under these conditions, hybridization can be induced by allowing the single strands of DNA form base pairs with complementary base sequences on another strand of DNA or RNA. During replication, repair and transcription, complex proteins (helicases during DNA replication, RNA polymerase during transcription) cause the separation of the two strands.

The cellular DNA and RNA polymerases of higher organisms are much more accurate than those of viruses because of their high specificities and, in the case of DNA polymerases, proofreading capabilities. This accounts for the high degree of mutation in viruses. However, this can have therapeutic advantages.

Ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) is a purine nucleoside analog exhibiting in vitro antiviral activity against a broad spectrum of DNA and RNA viruses. Ribavirin has shown clinical efficacy against both influenza A and B viruses. This antiviral activity is a result of the resemblance of this compound to nucleosides. Studies showed that ribavirin most closely resembles guanosine, as determined by x-ray crystallography (Figure 2-1). Once in cells, ribavirin is converted to its 5′-phosphate derivatives by cellular enzymes. The major metabolite is ribavirin-5′-triphosphate (RTP), and the intracellular concentration of the mono-, di-, and triphosphate derivatives probably is similar to that of other cellular nucleotides. Although not all polymerase/replication systems use ribavirin as they do guanosine, it has been shown that many of the systems that are inhibited by ribavirin are reversed by the addition of guanosine.

Until 2001, it was thought that the mechanism of action of ribavirin involved a decrease in cellular guanosine triphosphate (GTP) pools resulting from inhibition of inosine monophosphate dehydrogenase by ribavirin monophosphate. More recently, the mechanism of action for ribavirin has been expanded to include lethal mutagenesis of the viral genome as a result of ribavirin triphosphate utilization by the error-prone viral RNA-dependent RNA polymerase, and incorporation of ribavirin into viral RNA.

Figure 2-1. Comparison of the structures of ribavirin (1-β-D-ribofuranosyl- 1H-1,2,4-triazole-3-carboxamide) with the purine nucleoside guanosine.

Figure 2-2. The pseudo base (1,2,4-triazole 3-carboxamide) of ribavirin pairs

equivalently with cytosine and uracil. R denotes the polyribonucleotide strand.

In 2001 a critical study published in Nature Medicine showed in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus 3D pol. Ribavirin incorporation is mutagenic, because it serves as a template for incorporation of cytidine and uridine with equal efficiency (Figure 2-2). Ribavirin reduces infectious poliovirus production to as little as 0.00001 percent in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into “error catastrophe.” This study reveals a new concept about nucleoside analogs, that is, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.

RNA viruses like HIV and the influenza virus use a naturally high mutation rate of their RNA polymerases with low fidelity for replication to avoid and escape most treatments and vaccines. A RNA virus tends to mistakenly insert ribavirin into newly synthesized copies of their RNA genome. But ribavirin adds so many extra mutations to the virus during the replication of its genetic materials, it induces a flood of mutations and thus pushes the virus into a kind of genetic meltdown.

COMPREHENSION QUESTIONS

[2.1] Influenza virus is a class Vb virus, which means that it has a single (–)- stranded RNA for its genome. Which of the following best describes the immediate fate of this (–)-RNA when the virus enters the host cell?

A. It is used directly to encode viral proteins.

B. It is used as a template to synthesize a (+)-strand viral messenger

RNA (mRNA).

C. It is used as a template to synthesize viral DNA.

D. It is converted to a provirus.

E. It is integrated into the host cell genome.

[2.2] If a double-stranded DNA molecule undergoes two rounds of replication in an in vitro system that contains all of the necessary enzymes and nucleoside triphosphates that have been labeled with ³²P, which of the following best describes the distribution of radioactivity in the four resulting DNA molecules?

A. Exactly one of the molecules contains no radioactivity.

B. Exactly one of the molecules contains radioactivity in only one strand.

C. Two of the molecules contain radioactivity in both strands.

D. Three of the molecules contain radioactivity in both strands.

E. All four molecules contain radioactivity in only one strand. 

[2.3] A 48-year-old man has had a lengthy history of skin cancer. In the past 6 years he has had over 30 neoplasms removed from sun-exposed areas and has been diagnosed with xeroderma pigmentosum. Which of the following best describes the enzymatic defect in patients with xeroderma pigmentosum?

A. DNA polymerase α

B. DNA polymerase γ

C. DNA ligase

D. Excision repair enzymes

E. RNA polymerase III

Answers

[2.1] B. Class Vb viruses, since they have a minus single-stranded RNA genome, cannot use their genomic RNA directly to encode viral proteins. It is used to synthesize a (+)-stranded viral mRNA that is then used to encode the viral proteins. The influenza virus does this by the following mechanism: a virus-specific polymerase first cleaves an oligonucleotide from a host cell mRNA. It uses this as a primer that is elongated by the polymerase to synthesize the viral (+)-mRNA using the (−)-viral RNA genome as the template.

[2.2] C. After two rounds of replication using ³²P-labeled nucleoside triphosphates (NTPs), all four DNA molecules will be radioactive; two will be radioactive in both strands, two will be radioactive in only one strand.

[2.3] D. Xeroderma pigmentosum is a genetic disease in which the ability to remove pyrimidine dimers caused by exposure to UV light is impaired. The mechanism used to remove these pyrimidine dimers (also used to repair DNA that has formed adducts with carcinogenic compounds) is excision repair. The enzymes used in this repair mechanism cleave the affected strand on either side of damaged nucleotides. The oligonucleotide containing the damaged nucleotides is removed and the gap is filled in by DNA polymerase and DNA ligase.

BIOCHEMISTRY PEARLS

❖ The most stable DNA structure is formed when two polynucleotide chains are joined by hydrogen bonding between the side chain bases. The base pairing is specific in that adenine pairs with thymine and guanine pairs with cytosine (A-T; G-C)

❖ Experimentally, the double helix can be separated, or denatured, by increasing the temperature to well above 50°C (122°F); if the temperature is carefully decreased, renaturation occurs when the base pairs reform.

❖ Ribavirin is a purine nucleoside analog exhibiting in vitro antiviral activity against a broad spectrum of DNA and RNA viruses, including clinical efficacy against both influenza A and B virus.

❖ Ribavirin seems to cause mutagenic changes to RNA viruses, with ribavirin being inserted into newly synthesized copies of their RNA genome.

References

Crotty S, Maag D, Arnold JJ, et al. The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat Med 2001;6:1375–9. 

Gilbert BE, Knight V. Biochemistry and clinical applications of ribavirin. Antimicrob Agents Chemother 1986;30:201–5. 

Maag D, Castro C, Hong Z, et al. Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J Biol Chem 2001;276:46094–8.

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