Resistant Herpes Simplex Virus Research Articles

Resistance of herpes simplex virus to antiviral agents. Is it clinically important?

The deoxyguanosine analogue, acyclovir, represents a singularly important advance in antiviral therapy because it is an antiherpes drug which requires a viral-specific enzyme to form an active inhibitor of viral DNA replication. As discussed by Richards et al. in this issue of the Journal (p. 378), acyclovir is a preferred substrate for herpesvirus thymidine kinase (TK) which phosphorylates acyclovir to acyclovir monophosphate (Elion et aI., 1978). Cellular guanylate kinase then leads to the formation of acyclovir triphosphate (Miller and Miller, 1980). Acyclovir triphosphate is a substrate for the herpesvirus DNA polymerase; it is a competitive inhibitor for DNA polymerase activity, and appears to be a chain terminator of elongating herpesvirus DNA (Derse et aI., 1981; Furman et aI., 1979). Acyclovir triphosphate inhibits viral DNA polymerase 30 times more preferentially than any cellular polymerase (Elion et aI., 1978).

Enhanced mutagenesis parallels enhanced reactivation of herpes virus in a human cell line.

U.v. irradiation of human NB-E cells results in enhanced mutagenesis and enhanced reactivation of u.v.-irradiated H-1 virus grown in those cells ( Cornelis et al., 1982). This paper reports a similar study using herpes simplex virus (HSV) in NB-E cells. The mutation frequency of HSV (resistance of virus plaque formation to 40 micrograms/ml iododeoxycytidine ) increased approximately linearly with exposure of the virus to u.v. radiation. HSV grown in unirradiated cells gave a slope of 1.8 X 10(-5)m2/J, with 3.2 X 10(-5)m2/J for HSV grown in cells irradiated (3 J/m2) 24 h before infection. There was no evidence for mutagenesis of unirradiated virus by irradiated cells, as seen with H-1 virus. Enhanced reactivation of irradiated HSV in parallel cultures increased virus survival, manifested as a change in slope of the final component of the two-component survival curve from a D0 of 27 J/m2 in unirradiated cells to 45 J/m2 in irradiated cells. Thus, enhanced mutagenesis and enhanced reactivation occurred for irradiated HSV in NB-E cells. The difference in the enhanced mutagenesis of HSV (dependent on damaged DNA sites) and of H-1 virus (primarily independent of damaged DNA sites) is discussed in terms of differences in DNA polymerases.

Experimental skin infection with an acyclovir resistant herpes simplex virus mutant: response to antiviral treatment and protection against reinfection.

Skin infections induced in hairless mice with an Acyclovir resistant herpes simplex virus (HSV) mutant were not followed by the death of the animals, and the survivors had no evidence of latent infections in their sensory ganglia. However, mutant virus was detected in the ganglia during the acute phase of the infection. Mice inoculated with the mutant were fully protected against the fatal outcome of the infection when subsequently challenged with the relatively pathogenic parental virus. In addition the frequency of latent infections established after challenge was significantly reduced. Phosphonoacetic acid treatment of the primary mutant-induced infection abolished the protection against reinfection with parental virus. Acyclovir treatment of the primary infection with the mutant virus did not affect the protection against reinfection with parental virus. The results indicate that drug-resistant, latency-negative, HSV mutants are a promising starting point for the development of an attenuated HSV vaccine.

Resistance of herpes simplex virus to acycloguanosine—genetic and physical analysis

Resistance of herpes simplex viruses (HSV-1 and HSV-2) to acycloguanosine (ACG) is determined by the two genetic loci of HSV coding for deoxypyrimidine kinase (thymidine kinase) and DNA polymerase activity. Mutants of HSV-1 which induce defective deoxypyrimidine kinase (dPyK − mutants) activity can participate in interallelic complementation, and show variability in their resistance to acycloguanosine (ACG R). This allows for subdivision of dPyK − mutants based on their resistance to ACG. Mutants dPyK − and dPyK −11 are in one interallelic complementation group and both are 100-fold more resistant to ACG in a plaque reduction assay than is wild type herpes simplex virus type 1 (HSV-1). (The 50% inhibitory concentrations (ID 50) are 6.0 and 7.0 μM of ACG compared to 0.06 μM.) Mutant MDK (Kit) which cannot complement any other mutant is 500-fold more resistant to ACG (ID 50 = 50 μM). Other dPyK − mutants fall in between in their sensitivity to ACG. The map position of the mutations in the DNA polymerase locus which result in resistance to ACG ( acg r) was located by correlating the ACG sensitivity of HSV-1IHSV-2 intertypic combinants with restriction endonuclease analysis of their DNA. The mutations acg r and paa r (phosphonoacetic acid resistance) are contained in the same 1.3 kbp region of DNA (map units 40.2 to 41.0). The level of sensitivity to ACG is type specific, differing in HSV-1 and HSV-2. This type specificity is determined by DNA sequences within map units 39.6 to 41.0. Thus the DNA sequences for type-specific sensitivity and resistance to ACG are shown to be distinct and clustered in the same region as the HSV DNA polymerase locus.

Resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and DNA polymerase loci.

Acycloguanosine [9-(2-hydroxyethoxymethyl)guanine; acyclo-Guo] is a potent inhibitor of herpes simplex viruses (HSV); it is selectively phosphorylated in virus-infected cells. In order to define those viral functions that may mediate resistance to acyclo-Guo, the drug sensitivities of temperature-sensitive (ts) and phosphonoacetic acetic acid (PAA)-resistant mutants of HSV-1 and HSV-2 have been determined. Two distinct viral genetic loci are independently associated with acyclo-Guo resistance. Mutations resulting in diminished thymidine kinase activity are associated with resistance to inhibition by acyclo-Guo. Several PAA-resistant viruses that express wild-type levels of thymidine kinase activity are also resistant to acyclo-Guo. This suggests the importance of the viral DNA polymerase region in mediating acyclo-Guo resistance and is consistent with a close relationship between the PAAr mutation site and the AGGr locus. When wild-type HSV-1 is serially propagated under the selective pressure of acyclo-Guo, rapid emergence of resistant virus occurs, accompanied by the simultaneous appearance of thymidine kinase-deficient progeny.

In vitro and in vivo resistance of herpes simplex virus to 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine).

In vitro passage of the Patton strain of herpes simplex virus type 1 (HSV-1-P) in the presence of acycloguanosine (ACG) led to the emergence of a highly drug-resistant strain (HSV-1-P-ACG-R). Over 1% of virions in 6 of 15 HSV strains tested were able to form plaques in the presence of 10 mumol of ACG on first exposure to the drug. Therefore, there exists among HSV strains a broad range of susceptibilities to ACG, and some strains contain particles which are partially resistant to ACG before ever contacting the drug. HSV-1-P-ACG-R was partially resistant to iododeoxyuridine; conversely, iododeoxyuridine-resistant virus was highly resistant to ACG. ACG-resistant virus (HSV-1-P-ACG-R) was equal to its parent strain (HSV-1-P) in susceptibility to adenine arabinoside. The HSV-1-P-ACG-R strain produced corneal lesions in rabbits which were completely refractory to topical treatment with 1% unguent ACG, but responsive to 3% ACG. Partially resistant HSV could be isolated from the eyes of rabbits infected with ACG-susceptible virus and treated topically with either 1 or 3% ACG for 6 days.

In vitro RNA synthesis in nuclei from herpes simplex virus infected cells: Effects of ultraviolet-irradiated, defective and bromodeoxyuridine-resistant virus preparations

Transcription of virus DNA was detectable in vitro in nuclei isolated from cells infected with wild type and bromodeoxyuridine (BUdR)-resistant herpes simplex virus type 1 but was suppressed in nuclei of cells infected with ultraviolet irradiated or defective virus or cells treated with BUdR during infection.

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.