Natural killer (NK) cells are innate lymphocytes that are crucial for the control of infections and malignancies. NK cells express a variety of inhibitory and activating receptors that facilitate fine discrimination between damaged and healthy cells. Among them, a family of molecules that bind nectin and nectin-like proteins has recently emerged and has been shown to function as an important regulator of NK cell functions. These molecules include CD226, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), CD96, and cytotoxic and regulatory T cell molecule (CRTAM). In this Review, we focus on the recent advances in our understanding of how these receptors regulate NK cell biology and of their roles in pathologies such as cancer, infection and autoimmunity.

Balancing natural killer cell activation through paired receptors

Human herpesvirus 7 (HHV-7) is a recently isolated betaherpesvirus that is prevalent in the human population, with primary infection usually occurring in early childhood. HHV-7 is related to human herpesvirus 6 (HHV-6) in terms of both biological and, from limited prior DNA sequence analysis, genetic criteria. However, extensive analysis of the HHV-7 genome has not been reported, and the precise phylogenetic relationship of HHV-7 to the other human betaherpesviruses HHV-6 and human cytomegalovirus has not been determined. Here I report on the determination and analysis of the complete DNA sequence of HHV-7 strain JI. The data establish that the close biological relationship of HHV-6 and HHV-7 is reflected at the genetic level, where there is a very high degree of conservation of genetic content and encoded amino acid sequences. The data also delineate loci of divergence between the HHV-6 and HHV-7 genomes, which occur at the genome terminal in the region of the terminal direct-repeat elements and within limited regions of the unique component. Of potential significance with respect to biological and evolutionary divergence of HHV-6 and HHV-7 are notable structural differences in putative transcriptional regulatory genes specified by the direct-repeat and immediate-early region A loci of these viruses and the absence of an equivalent of the HHV-6 adeno-associated virus type 2 rep gene homolog in HHV-7.

Determination and analysis of the complete nucleotide sequence of human herpesvirus.

Ribonucleotide reductase is an essential enzyme for DNA synthesis in all prokaryotic and eukaryotic cells; it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. Several herpesviruses including herpes simplex virus type 1 (HSV-1)1, HSV-22, pseudorabies virus (PRV)3, equine herpesvirus type 1 (EHV-1)4 and Epstein–Barr virus (EBV)5 have been found to induce novel ribonucleotide reductase activities. There is evidence that the HSV-1 ribonucleotide reductase activity is virus-encoded6 and essential for virus replication7. This makes herpesvirus ribonucleotide reductases potential targets for antiviral chemotherapy. The HSV-1-encoded enzyme consists of two summits8,9,18: V136, the large subunit of relative molecular mass (Mr) 136,000 (136K) (RR1), which has been shown to be essential for enzyme activity7, and V38, the small subunit (RR2) which forms a complex with the large subunit and is also likely to be essential for enzyme activity9. Two particular features of the enzyme make it an attractive antiviral target. First, there is evidence for a common, highly conserved herpesvirus ribonucleotide reductase and second, the interaction between the large and small subunits may itself be exploitable. Here we identify a synthetic peptide which specifically inhibits the activity of virus-induced enzyme. We deduce that the mechanism of inhibition involves interference with the normal interaction between the two types of subunit.

Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides

Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells. The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies. Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref. 12) and subunit 2 (ref. 13) of the Escherichia coli and mammalian enzymes. To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C. et al., in preparation). We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits. This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions.

Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2.

Summary
The role of the herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR) in the pathogenicity of the virus has been examined by use of mutants with lesions in either the large or small subunit of the enzyme. The virulence of the mutants in mice was reduced by about 106-fold when compared with that of the parental virus (HSV type 1 strain 17), while the virulence of a revertant of one of the mutants was restored to within about 100-fold of that of the parent virus. These experiments demonstrate that activity of the HSV RR is essential for virus pathogenicity in mice and suggests that the enzyme is a valid target for specific antiviral compounds.

Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target.

Summary
In nearly all systems studied, ribonucleotide reductase consists of two non-identical subunits. We present here the results of our study on herpes simplex virus (HSV) ribonucleotide reductase in favour of the existence of two subunits, H1 and H2, different from the mammalian subunits, M1 and M2. First, although the viral subunits could not be separated by Blue Sepharose chromatography (unlike mammalian subunits), they seemed to dissociate at very low protein concentration as suggested by the non-linear relationship between activity and low protein concentration. Second, pyridoxal phosphate (Pyr.P)-NaBH4 treatment and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) treatment of partially purified extract of mammalian ribonucleotide reductase which inactivated M1 and M2 respectively also inhibited the HSV ribonucleotide reductase. This activity could be restored by mixing Pyr.P-NaBH4-treated extracts with MAIQ-treated extracts of viral ribonucleotide reductase, suggesting that each treated extract contains one active subunit. Moreover, the addition of exogenous M1 and M2 subunits to one or the other of these two treated extracts did not produce any detectable reductase activity. Our interpretation of these results is that the two subunits H1 and H2 which could dissociate upon treatment did not form enzymically active hybrids with the mammalian subunits. Also, the higher degree of resistance to heat inactivation and to hydroxyurea of the viral reductase as compared to the mammalian enzyme suggests that H1 differs from M1 and H2 from M2.

Herpes Simplex Virus Ribonucleotide Reductase Induced in Infected BHK-21/C13 Cells: Biochemical Evidence for the Existence of Two Non-identical Subunits, H1 and H2

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4+ T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Disclosed herein are antiviral peptides of the formula A-R⁸-R⁹-R¹⁰-R¹¹-R¹²-R¹³-R¹⁴-R¹⁵-B wherein A is from zero up to seven amino acid residues and includes a terminal hydrogen or a terminal N-acyl, or A is a phenylacetyl with optional substitution of the para position of the phenyl, R⁸, R⁹, R¹⁰, R¹³, R¹⁴ and R¹⁵ are various amino acid residues with the stipulation that one or more of the four amino acid residues immediately preceeding Rʺ may optionally be deleted, R¹¹ and R¹² are independently Val, D-Val, Nva, D-Nva, Leu, D-Leu, Nle, D-Nle, Ile or D-Ile, and B is hydroxy, amino or lower alkylamino. The antiviral activity of the peptides can be enhanced by combining them with a protease inhibitor. The peptides and the combination are useful for the treatment of herpes viral infections in mammals.

Antiviral peptides and means for treating herpes infections

Ribonucleotide reductase from mammalian cells is composed of two nonidentical subunits M1 and M2 which are both required to form the catalytic site. The level of ribonucleotide reductase activity i...

Eric A. Cohen

Papers

Herpes Simplex Virus Ribonucleotide Reductase Induced in Infected BHK-21/C13 Cells: Biochemical Evidence for the Existence of Two Non-identical Subunits, H1 and H2

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Antiviral peptides and means for treating herpes infections

Activities of both ribonucleotide reductase subunits, M1 and M2, decrease upon serum starvation of baby hamster kidney 21/C13 cells

Peptides antiviraux et moyens pour le traitement de l'herpès