Thomas R. Jones

Novel Class of Thiourea Compounds That Inhibit Herpes Simplex Virus Type 1 DNA Cleavage and Encapsidation: Resistance Maps to the UL6 Gene

ABSTRACT In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered.N-{4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl}-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.

An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

ABSTRACT The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

Identification of Small Molecule Compounds That Selectively Inhibit Varicella-Zoster Virus Replication

ABSTRACT A series of nonnucleoside, N-α-methylbenzyl-N′-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.

Specious hybridization between herpes simplex virus DNA and human cellular DNA.

Apparent hybridization between human cellular DNA and herpes simplex virus DNA was blocked by the presence of guanine-rich ribo- and deoxyribonucleic acid polymers. The data indicate that the apparent hybridization may very well be artifactual and not represent long stretches of authentic base sequence homology.

Specific Inhibition of Human Cytomegalovirus Glycoprotein B-Mediated Fusion by a Novel Thiourea Small Molecule

ABSTRACT A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.

Thiourea inhibitors of herpes viruses. Part 1: bis-(aryl)thiourea inhibitors of CMV.

Bis-(aryl)thioureas were found to be potent and selective inhibitors of cytomegalovirus (CMV) in cultured HFF cells. Of these, the thiazole analogue 38 was investigated as a potential development candidate.

Hybridization of herpes simplex virus dna and human ribosomal DNA and RNA

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.

A simple repetitive sequence common to herpes simplex virus type 1 and human ribosomal DNAs

The simple sequence GGC was tandemly repeated in herpes simplex virus type 1 DNA and human 28S rDNA and its mature 28 S rRNA transcript. The sequence homology was responsible for the observed hybridization between the two DNAs under high stringency blot hybridization conditions.

Hybridization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC]n and heterodisperse cellular DNA and RNA

A small DNA fragment containing the simple sequence [GGC]10 from the long repeat of herpes simplex virus type 1 (HSV-1) DNA hybridized to cellular DNA and polyadenylated RNA from different mammalian species. The number and intensity of blot hybridization signals were increased in human compared with rodent and simian nucleic acids. The hybridization was blocked specifically by human 28S ribosomal DNA, which shares only the GGC repeats with the herpes simplex virus DNA. These data indicate that GGC repeats were common components of cellular DNA and were expressed in mRNA. Blot hybridization analysis of viral RNA from the HSV-1 gene regions encompassing the GGC repeats revealed abundant stable mRNAs from portions of the virus genome not previously analyzed in detail and indicated that the viral GGC sequence was not expressed in stable cytoplasmic mRNA.

Heteroduplex analysis of cloned fragments of herpes simplex virus DNAs

Abstract The heteroduplex map of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNAs was constructed with recombinant HSV-1 and HSV-2 DNAs so that the overall map was built up piece by piece across the virus genome. Extensive regions of good base sequence homology and some regions of partial homology were mapped. In certain cases, known virus functions were assigned to specific positions on the HSV-1-HSV-2 DNA heteroduplex map, and the sequences of HSV-1 and HSV-2 genes were compared.