(D)- and (L)-cyclohexenyl-G, a new class of antiviral agents: synthesis, conformational analysis, molecular modeling, and biological activity

(D)- and (L)-cyclohexeneyl-G were synthesized enantioselectively starting from (R)-carvone. Both show potent and selective anti-herpesvirus activity (HSV-1, HSV-2, VZV, CMV). Molecular modeling demonstrates that both isomers are bound in the active site of HSV-1 thymidine kinase in a high-energy conformation with the base moiety orienting in an equatorial position. It is believed that the flexibility of the cyclohexene ring is essential for their antiviral activity.     

SYNTHESIS AND IN VITRO ACTIVITY OF D- AND L-ENANTIOMERS OF 5-(TRIFLUOROMETHYL)URACIL NUCLEOSIDE DERIVATIVES

Recently, β-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF₃-β-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF₃-β-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).     

Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG) against Herpes simplex virus type 1 infection in vitro

Pentagalloylglucose (PGG) is a natural polyphenolic compound with broad-spectrum anti-viral activity, however, the mechanisms underlying anti-viral activity remain undefined. In this study, we investigated the effects of PGG on anti-viral activity against Herpes simplex virus type 1 (HSV-1) associated with autophagy. We found that the PGG anti-HSV-1 activity was impaired significantly in MEF-atg7^–/– cells (autophagy-defective cells) derived from an atg7^–/– knockout mouse. Transmission electron microscopy revealed that PGG-induced autophagosomes engulfed HSV-1 virions. The mTOR signaling pathway, an essential pathway for the regulation of autophagy, was found to be suppressed following PGG treatment. Data presented in this report demonstrated for the first time that autophagy induced following PGG treatment contributed to its anti-HSV activity in vitro.     

LIPOSOME-INCORPORATED SANTOLINA INSULARIS ESSENTIAL OIL: PREPARATION,CHARACTERIZATION AND IN VITRO ANTIVIRAL ACTIVITY

The effect of liposomal inclusion on the stability and in vitro antiherpetic activity of Santolina insularis essential oil was investigated. In order to study the influence of vesicle structure on the liposome properties, multilamellar and unilamellar vesicles were prepared by the film method and sonication, respectively. Vesicles were obtained from hydrogenated soya phosphatydilcholine and cholesterol. Formulations were examined for their stability for over one year monitoring the drug leakage from vesicles and the average size distribution. The stability of the incorporated oil was verified by studying its quali-quantitative composition. The antiviral activity was studied against Herpes simplex virus type 1 (HSV-1) by plaque reduction and yield reduction assays. Results showed that Santolina insularis essential oil can be incorporated in high amounts in the prepared liposomes, which successfully prevented its degradation. Moreover, stability studies pointed out that vesicle dispersions were stable for at least one year and neither oil leakage nor vesicle size alteration occurred during this period. Antiviral activity assays demonstrated that Santolina insularis essential oil is effective in inactivating HSV-1 and that the activity is principally due to direct virucidal effects. Free essential oil proved to be more effective than liposomal oil and a different activity was discovered which related to the vesicular structure. The ED₅₀ values, significantly lower when cells were pre-incubated with the essential oil before the virus adsorption, indicate an intracellular mechanism in the antiviral activity of Santolina insularis. Moreover, liposomal Santolina essential oil is non toxic in the range of the concentration tested.     

Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157-7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.     

Mutational analysis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): characterization of HSV type 1 (HSV-1)/HSV-2 chimeras.

During lytic herpes simplex virus (HSV) infections, the half-lives of host and viral mRNAs are regulated by the HSV virion host shutoff (Vhs) protein (UL41). The sequences of the UL41 polypeptides of HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical. In spite of this similarity, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. To examine type-specific differences in Vhs function, we compared the Vhs activities of UL41 alleles from HSV-1(KOS) and HSV-2(333) by assaying the ability of a transfected UL41 allele to inhibit expression of a cotransfected reporter gene. Both HSV-1 and HSV-2 alleles inhibited reporter gene expression over a range of vhs DNA concentrations. However, 40-fold less of the HSV-2 allele was required to yield the same level of inhibition as HSV-1, indicating that it is significantly more potent. Examination of chimeric UL41 alleles containing various combinations of HSV-1 and HSV-2 sequences identified three regions of the 333 polypeptide which increase the activity of KOS when substituted for the corresponding amino acids of the KOS protein. These are separated by two regions which have no effect on KOS activity, even though they contain 43 of the 74 amino acid differences between the parental alleles. In addition, alleles encoding a full-length KOS polypeptide with a 32-amino-acid N-terminal extension retain considerable activity. The results begin to identify which amino acid differences are responsible for type-specific differences in Vhs activity.     

Immunization with a dominant-negative recombinant Herpes Simplex Virus (HSV) type 1 protects against HSV-2 genital disease in guinea pigs

Background  

CJ9-gD is a novel dominant-negative recombinant herpes simplex virus type 1 (HSV-1) that is completely replication-defective, cannot establish detectable latent infection in vivo, and expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In the present study, CJ9-gD was evaluated as a vaccine against HSV-2 genital infection in guinea pigs.     

Structural features of nectin-2 (HveB) required for herpes simplex virus entry

One step in the process of herpes simplex virus (HSV) entry into cells is the binding of viral glycoprotein D (gD) to a cellular receptor. Human nectin-2 (also known as HveB and Prr2), a member of the immunoglobulin (Ig) superfamily, serves as a gD receptor for the entry of HSV-2, variant forms of HSV-1 that have amino acid substitutions at position 25 or 27 of gD (for example, HSV-1/Rid), and porcine pseudorabies virus (PRV). The gD binding region of nectin-2 is believed to be localized to the N-terminal variable-like (V) Ig domain. In order to identify specific amino acid sequences in nectin-2 that are important for HSV entry activity, chimeric molecules were constructed by exchange of sequences between human nectin-2 and its mouse homolog, mouse nectin-2, which mediates entry of PRV but not HSV-1 or HSV-2. The nectin-2 chimeric molecules were expressed in Chinese hamster ovary cells, which normally lack a gD receptor, and tested for cell surface expression and viral entry activity. As expected, chimeric molecules containing the V domain of human nectin-2 exhibited HSV entry activity. Replacement of either of two small regions in the V domain of mouse nectin-2 with amino acids from the equivalent positions in human nectin-2 (amino acids 75 to 81 or 89) transferred HSV-1/Rid entry activity to mouse nectin-2. The resulting chimeras also exhibited enhanced HSV-2 entry activity and gained the ability to mediate wild-type HSV-1 entry. Replacement of amino acid 89 of human nectin-2 with the corresponding mouse amino acid (M89F) eliminated HSV entry activity. These results identify two different amino acid sequences, predicted to lie adjacent to the C' and C" beta-strands of the V domain, that are critical for HSV entry activity. This region is homologous to the human immunodeficiency virus binding region of CD4 and to the poliovirus binding region of CD155.     

Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol,Fusarenon-X,and Nivalenol) against Herpes Simplex Virus Types 1 and 2

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC₅₀) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.     

Synthesis and antiviral activity of 1-(1,3-disubstitutedimidazolidyn-2-ylidene)-3-ethoxycarbonylmethylurea derivatives

Novel 1-(1,3-disubstituted-imidazolidyn-2-ylidene)-3-ethoxycarbonylmethylurea derivatives (3a–3j) were obtained from appropriate 1-aryl-3-arylsulfonyl-1H-imidazolidine-2-imines (1a–1j) and ethyl isocyanatoacetate (2), which were subjected to condensation. Seven compounds were tested for their antiviral activity against HSV-1 and CVB3 viruses. Among the tested compounds, 3c was found to be active against HSV-1, proving that 4-methoxy substituent as R and 4-methyl substituent as R₁ are most beneficial for activity against this virus. Furthermore, 3e and 3g were active against CVB3, which demonstrated that both 4-methyl and 4-chloro substitution is tolerated as R₁, whereas 4-chloro and 2-methoxy substituents are best as R. It was also shown that the active compounds are characterized by relatively big surface area, small ovality, and greatest HOMO and LUMO energies in comparison to the rest of the compounds.     

Mothers of children with Down syndrome have higher herpes simplex virus type 2 (HSV-2) antibody levels

Summary The antibody response to herpes simplex virus (HSV) was studied in 53 mothers of children with Down syndrome (Ds) and compared with that in 154 controls, using sera sampled during pregnancy or at delivery. Conventional analysis of HSV complement fixing antibodies showed the same frequency of positivity for the two groups (70%). When the levels of IgG antibodies to an HSV-1 and an HSV-2 antigen preparation were determined by an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the Ds and control mothers had similar levels of IgG antibodies to HSV-1, whereas the level of IgG antibodies to HSV-2 was significantly (P<0.001) higher in Ds mothers. The ratio of HSV-2 to HSV-1 ELISA IgG was calculated for each mother and the distribution of these ratios also differed significantly between the control and Ds mothers. The differences found were not due to differences in age distribution in the control and Ds groups. For comparison a third procedure, measurement of thymidine kinase blocking antibody (TK ab), was used. With this procedure the mothers were divided into groups estimated to be positive for HSV-1, HSV-2, or both. Statistical analyses showed a good correlation between the type found in TK ab analyses and the ratio found in the ELISA HSV test. The results clearly demonstrated an overrepresentation of HSV-2 antibody positivity among Ds mothers, though not of sufficient magnitude to imply that HSV-2 can be the major cause of Ds. It is discussed whether HSV-2 might be related to the recently increased birthrate of children with Ds among young mothers in Sweden or to localized geographical clustering of Ds births, or whether the increased HSV-2 antibody positivity merely indicates that factors following the same epidemiological pattern are involved in the aetiology of Ds.     

Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Synthesis and biological activity of tricyclic analogues of 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine

The base moiety of the potent antiherpetic agent 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine 3 was transformed into that of the tricyclic 3,9-dihydro-9-oxo-6-R-5H-imidazo[1,2-a]purine system. The tricyclic analogues 5a-d were evaluated for their activity against herpes viruses as well as for cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumor cells. Marked activity was found against VZV. The 6-phenyl-substituted fluorescent analogues 5c and d were comparable to that of parent 3 in activity against the VZV strain YS and were 3-fold less active against the VZV strain OKA. The compounds 5a-d also showed marked activity against HSV-1 (KOS) and HSV-2 (G)-against the former generally approximately comparable to that of acyclovir 1a and one order of magnitude lower than 3; against the latter comparable to that of 1a and approximately 6- to 30-fold lower than that of 3. The most pronounced cytostatic activity (5-fold lower than that of 3) was exhibited by compounds 5c and d. Tricyclic analogues with pseudosugar moieties are intrinsically bio-active.     

Modifications on the heterocyclic base of ganciclovir,penciclovir, acyclovir - syntheses and antiviral properties

АBSTRACT

Esters of the antiherpetic drugs ganciclovir, penciclovir with the bile acids (cholic, chenodeoxycholic and deoxycholic) and amino acid esters of acyclovir were generated and evaluated for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The antiviral assays demonstrated that modified analogs of ACV and PCV are less active compared to the initial substances against HSV-1and HSV-2. CC50 for ganciclovir-deoxycholate corresponded to the CC50 of the other analogs and its activity is lower than ganciclovir. Obtained results show that tested modification do not improve bioavailability of nucleoside analogs in cells.     

Synthesis and Biological Evaluation of 9-(f-2, c-3-Bishydroxymethyl-r-cyclopropylmethyl)-9H-adenine (A Lower Methylene Homolog of Carbocyclic Oxetanocin) and Related Compounds

Abstract

Adenine (7 and 16), thymine (9a and 18a), and 5-fluorouracil (9b and 18b) involving f-2, c-3-bishydroxymethyl-r-1-cyclopropylmethyl- and t-2 t-3-bishydroxymethyl-r-1-cyclopropylmethyl residues were synthesized, starting from trans-1, 4-dibenzyloxy-2-butene and its cis isomer, respectively. These compounds were evaluated for anti HSV-1 activity.     

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1)

Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy. HIGHLIGHTS: ?HSV-1 activates glycolysis by PFK-1 activation. ?In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ?PFK-1 knockdown impairs HSV-1 replication. ?HSV-1-mediated glycolysis activation increases ATP content.     

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-β1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-β1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.     

Binding of HSV-1 Glycoprotein K (gK) to Signal Peptide Peptidase (SPP) Is Required for Virus Infectivity

Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.     

Synthesis and Antiviral Activities of Enantiomeric 1-[2-(Hydroxymethyl) Cyclopropyl] Methyl Nucleosides

Abstract

Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100μM.