Problems

PART

1 The drug acycloguanosine, sold as acyclovir, has been one of the most successful antiviral compounds produced. Acyclovir is used in the treatment of herpes simplex virus infections. These viruses replicate their double-stranded DNA genomes using a virus-specific DNA polymerase. The structure of acyclovir is show below:

1 yOv CH2

1 yOv CH2

  • a) Given the structure of this drug, what is the specific effect of this nucleoside analogue on herpes virus DNA replication? Your answer should refer to a particular structural feature of the drug.
  • b) Acyclovir is administered to patients in the form shown. What must happen to this drug inside the cell before it can inhibit viral DNA replication? Again, your answer must refer to a particular structural feature of the drug.

2 You have prepared two highly purified suspensions ofpoliovirus, each grown on a different host cell. The first (stock A) was grown on HeLa cells (a human cell line). The second (stock B) was grown on AGMK cells (a monkey kidney cell line). Each suspension has a total volume of 10.0 ml. The virus stocks were titered by diluting the stocks and performing plaque assays. Dilutions were made by taking 1 part of the virus stock and mixing it with 9999 parts of buffer (dilution 1). A further 1 to 104 dilution was made, using this same procedure. The resulting suspension (dilution 2) was plaque assayed in duplicate on a lawn ofsusceptible cells. In addition, you measured the optical density ofthe original virus stocks at a wavelength of 260 nm. You know that an optical density (OD260) of 1.0 equals 1013 poliovirus particles/ml. The data you obtained are shown in the following table.

Virus stock

Host cell

Plaques in 1 ml of dilution 2 (replicate plates)

OD260 of virus stock

A

Hela

190 and 210

0.1

B

AGMK

48 and 52

0.5

(a) What are the plaque-forming unit (PFU) to particle ratios for the two viral stocks?

Stock B =_

  • b) Which host cell line produced the most total virus particles?
  • c) Which host cell line produced the most total infectious virus?

3 The Svedberg equation that describes the motion of a molecule through a solution under the influence of a centrifugal field is:

s = = M (1-VPsol ) ®2r N„f where 5, the Svedberg coefficient, is a function of the molecular weight (M) and the frictional coefficient (f). The constants in the equation are Avagadro's number (Nav), the partial specific volume of the molecule (v), and the density of the solution (psol). The table below gives some relevant data for several DNA molecules:

DNA

Molecular weight

Configuration3

PBR322 DNA

2.84 X 106

ds, circularb

PBR322 DNA digested with EcoRIc

2.84 X 106

ds, linear

Phage T4 DNA

1.12 X 108

ds, linear

Phage T7 DNA

2.5 X 107

ds, linear

Phage OX1s74 RF DNA

3.76 X 106

a Assume that fdsJmear >> fds.circular-

b Assume the same degree of supercoiling for all of the circular molecules. c pBR322 has only one recognition site of EcoRI.

Notes:

a Assume that fdsJmear >> fds.circular-

b Assume the same degree of supercoiling for all of the circular molecules. c pBR322 has only one recognition site of EcoRI.

Predict the sedimentation behavior (sedimentation rate) of the following pairs of molecules. In each case, state whether the indicated molecule of the pair will move "faster" or "slower" relative to the other member of the pair. (Note: you do not need to calculate a sedimentation rate. You need to determine the relative behavior of the pair of molecules in each case.)

Molecules

Relative sedimentation rate

1

2

1

2

Phage T4 DNA

Phage T7 DNA

pBR322 DNA

Phage OX174 RF DNA

pBR322 DNA

pBR322 DNA digested with EcoRI

Phage T7 DNA

pBR322 DNA digested with EcoRI

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