Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon.

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (alpha-IFN). Using the colorimetric method, HSV isolates had ED50s ranging from 0.03 +/- 0.02 micrograms/ml to 0.164 +/- 0.03 micrograms/ml for ACV and 6.3 +/- 5.2 IU/ml to 55.0 +/- 11.4 IU/ml for alpha-IFN. With the DNA hybridization method, ED50s ranged from 0.033 +/- 0.012 micrograms/ml to 0.190 +/- 0.031 micrograms/ml for ACV and 8.5 +/- 5.0 IU/ml to 43.5 +/- 6.0 IU/ml for alpha-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (greater than 50.0 micrograms/ml). The values obtained by the two methods showed good correlation (r = 0.724, P = 0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.

Selection for spontaneous null mutations in a chromosomally-integrated HSV-1 thymidine kinase gene yields deletions and a mutation caused by intragenic illegitimate recombination.

Spontaneous null mutations represent low frequency events that irreversibly and completely inactivate a gene, and can often consist of major gene alterations. To study the molecular mechanisms leading to recessive spontaneous null mutations in the human genome, we designed and tested a selection procedure in cell culture to enrich for this rare class of spontaneous mutations. The KT cell line contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene and the neomycin-resistance gene (neo), from plasmid pSV2neoKT, integrated as a single-copy in the human tk- cell line 143B. The HSV-1 tk gene was the target for spontaneous gene inactivation, and antiviral drugs (acyclovir, trifluorothymidine and ganciclovir) were used, in combination, to provide a selective enrichment for null mutations over the background of more frequent and revertible point mutations. The tk- mutations obtained with this multiple drug selection assay appeared at a very low frequency, rarely reverted to wild-type (tk+), and the TK protein was observed only in 4.8% of these null mutants. Deletions of the entire tk gene, or its 3' region, constituted the major class of DNA rearrangements seen in the null mutations. Additionally, one of the null mutants contained an intragenic 106-bp duplication within a 43-bp deleted region of the tk gene. We propose this mutation to be the outcome of an intragenic gene conversion event which may have been facilitated by short regions of junctional homology.

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein.

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.