Jennie G. Jacobson

A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells.

Herpes simplex virus encodes a ribonucleotide reductase that is not essential for virus growth in dividing cells at 37 degrees. This enzyme has been proposed as a target for antiviral drugs; its utility in this regard could depend upon its importance in vivo. To test the requirement of viral ribonucleotide reductase in a mammalian host, we tested a mutant virus, lacking most of the gene encoding the ribonucleotide reductase large subunit, in a mouse eye model of pathogenesis and latency where the wild-type virus establishes reactivatable latent infections in trigeminal ganglia following corneal inoculation. The deletion mutant was severely impaired in its ability to replicate acutely in the eye and in the trigeminal ganglion and failed to establish reactivatable latent infections. In contrast, a recombinant virus in which the deleted sequences were restored was competent for both acute and latent infections. The defects of the deletion mutant in the mouse may be related to its severely impaired growth at 38 degrees in mouse cells relative to its growth in Vero cells. These results indicate that ribonucleotide reductase is critical for productive acute and reactivatable latent infections in mice and replication in mouse cells at 38 degrees and suggest that caution be exercised in extrapolating from studies conducted in mice to human infections when judging the utility of this enzyme as a target for antiviral chemotherapy.

Very late activation antigens on rheumatoid synovial fluid T lymphocytes. Association with stages of T cell activation.

Lymphocytes from the synovial fluid of eight out of eight rheumatoid arthritis (RA) patients had elevated very late activation antigen-1 (VLA-1) expression (10-36% positive cells), whereas peripheral blood lymphocytes (PBL) from RA patients and healthy controls had low VLA-1 expression (0-6% positive cells). During 1-2 wk of in vitro culture, VLA-1 increased on synovial fluid cells but remained low on PBL. In comparison, the interleukin 2 receptor (IL-2 R) was less prominent than VLA-1 on fresh synovial fluid cells, did not increase on cultured synovial fluid T cells, but did increase greatly on cultured PBL. The mitogen PHA reversed or prevented the appearance of VLA-1+, IL-2 R- synovial fluid cells during in vitro culture, thus giving IL-2 R+, VLA-1- cells. These results emphasize that VLA-1+ SF cells are different from resting cells or IL-2 R+ activated PBL T cells, and VLA-1 on synovial fluid T cells may be incompatible with mitogen stimulation. In addition, the VLA-2 heterodimer (165,000/130,000 relative molecular mass [Mr]) was regulated opposite to the VLA-1 heterodimer (130,000/210,000 Mr) on synovial lymphocytes, and thus the VLA-1/VLA-2 ratio is another indicator of the stage of T cell activation.

Low levels of herpes simplex virus thymidine-thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model☆

Herpes simplex virus mutant KG111 contains a nonsense mutation at codon 44 of the viral thymidine kinase (tk) gene and produces low amounts of a truncated tk polypeptide. We tested mutant KG111 and related viruses that specify varying amounts of similar truncated tk polypeptides for their sensitivities to antiviral nucleoside analogs at different temperatures using plaque reduction assays. The results of these assays showed that the nonsense mutation confers high resistance to bromovinyldeoxyuridine (BVdU) at any temperature and temperature-dependent resistance to acyclovir (ACV), buciclovir (BCV), ganciclovir (DHPG), and fluoroiodoarabinouracil (FIAU). Above relatively low threshold levels of tk that varied depending on the drug tested, viruses exhibited full sensitivity to ACV, BCV, DHPG, and FIAU at 34 degrees. Below these threshold levels, however, decreases in drug sensitivity were linear with decreases in tk levels, forming the basis of a pharmacological assay for tk gene expression. Studies of thymidine (TdR) anabolism in infected 143 tk-cells showed that when high TdR concentrations were added to the medium, KG111 directed thymidine monophosphate (TMP) formation at rates consonant with the amount of tk polypeptide produced by the mutant. When low concentrations to TdR were added to the medium, however, KG111 directed TMP formation at a rate similar to that directed by wild-type virus, indicating that the truncation of the tk polypeptide had little or no effect on tk activity at 34 degrees. Subsequent anabolism to thymidine diphosphate and thymidine triphosphate was reduced in KG111-infected cells, indicating a defect in TMP kinase activity that explains this mutants resistance to BVdU. Despite the low levels of tk and TMP kinase activity expressed by KG111, this mutant established reactivatable latent infections as efficiently as wild-type virus in a mouse model.

Synergistic effects on ganglionic herpes simplex virus infections by mutations or drugs that inhibit the viral polymerase and thymidine kinase

Herpes simplex virus encodes proteins, such as DNA polymerase, that are essential for its replication and proteins, such as thymidine kinase, that are not essential for replication in cell culture, but are important for pathogenesis in animal models. However, certain mutations affecting these proteins exert little or no effect on replication or pathogenesis. We tested the effects of combining two such mutations--one that alters DNA polymerase and one that decreases but does not abolish thymidine kinase activity--on replication in cultured cells and on acute and latent infections in mice. The double mutant replicated similarly to the single mutants and wild-type virus both in cell culture and acutely in the mouse eye. However, it was severely impaired for acute replication in trigeminal ganglia and for reactivatable latent infections. This impairment depended upon the polymerase mutation. Similarly, although Ro 31-5140, a thymidine kinase inhibitor, did not potentiate the antiviral effects of phosphonoacetic acid, a polymerase inhibitor, in cell culture, the two drugs in combination substantially inhibited viral reactivation from latency at concentrations that had little or no effect when used singly. These synergistic effects may have implications for viral functions during pathogenesis and for antiviral chemotherapy.