In vitro fermentations of pure cellulose by mixed ruminal microorganisms were conducted under conditions in which pH declined within ranges similar to those observed in the rumen. At low cellulose concentrations (12.5 g/L), the first-order rate constants (k) of cellulose disappearance were successively lower at initial pH values of 6.86, 6.56, and 6.02, but in each case the value of k was maintained over a pH range of 0.3 to 1.2 units, as the fermentation progressed. Plots of k versus initial pH were linear, and k displayed a relative decrease of approximately 7% per 0.1 unit decrease in pH. At high cellulose concentration (50 g/L) and an initial pH of 6.8, cellulose digestion was initially zero order, the absolute rate of digestion declined with pH and digestion essentially ceased at pH 5.3 after only 30% of the added cellulose was digested. Further incubation resulted in a loss of bound N and P, suggesting that at low pH cells lysed or detached from the undigested fibers. Pure cultures of ruminal cellulolytic bacteria also were able to ferment cellulose to a minimum pH of 5.1 to 5.3, but the extent of fermentation was increased by coculture with noncellulolytic bacteria. A model is proposed in which the first-order rate constant of cellulose digestion is determined by the pH at which the fermentation is initiated, and end product ratios reflect greater activity of the noncellulolytic population as pH declines.

The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 M. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC 50 s) below 20 M were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC 50 , ϳ12 M). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC 50 , ϳ18 M), protected cells from infection (EC 50 , ϳ72 M), and inactivated virions in solution (EC 50 , ϳ138 M). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that primarily causes mucocutaneous ulcers. Less common but more serious infections can result in encephalitis, blinding keratitis, or neonatal herpes (78). Several antivirals are approved for the treatment of HSV infections but are ineffective in certain situations and do not eliminate latent infections (20,56,65). Thus, new strategies are needed to deal with these infections.HSV-1 infection is initiated by the binding of glycoprotein C (gC) to cell surface heparan sulfate (69,71,72). The HSV-1 gB protein (gB-1) can also bind to heparan sulfate proteoglycan, but the binding is less efficient (37). Following attachment, gD binds to any of four cellular coreceptors, including HVEM (herpesvirus entry mediator), nectin 1, nectin 2, or 3-O-sulfated heparan sulfate, resulting in a conformational change in gD (18,28,41,50,51). The conformational change in gD is then thought to trigger the formation of the fusion complex, which includes gB and the gH-gL heterodimer. The coexpression of gD, gB, and gH-gL in the same cell results in cell-cell fusion, indicating that these four proteins constitute the minimal fusion apparatus (10, 24, 52, 61, 75). It has been suggested that gH-gL and gB are recruited to gD independently of one another and possibly interact with each other (3). Recent studies show that HSV-1 fusion is mediated through a hemifusion intermediate involving gH-gL but that the complete fusion event requires the action of gB (73). gB-1 interacts with the cell surface (7,59,60), and a cell surface...

RC-2 was active against HSV-1 KOS in cultures and showed protective activity in vivo when used in a prophylactic mode, but the peptide showed limited activity in a postinfection herpes keratitis model. These findings support data obtained from experiments with HIV-1, HSV-2, and influenza A, indicating that RCs inhibit the entry of viruses rather than their replication.

Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 microM), bHOMd (up to 200 microM), or TAT-C (up to 400 microM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TATC. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC50 values of 70 and 93 microM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 microM, although cell proliferation was blocked at concentrations of EB above 50 microM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC50 values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.

Previously, it was shown that cationic ␣-peptides derived from the human immunodeficiency virus TAT protein transduction domain blocked herpes simplex virus type 1 (HSV-1) entry. We now show that cationic oligomers of ␤-amino acids ("␤-peptides") inhibit HSV-1 infection. Among three cationic ␤-peptides tested, the most effective inhibition was observed for the one with a strong propensity to adopt a helical conformation in which cationic and hydrophobic residues are segregated from one another ("globally amphiphilic helix"). The antiviral effect was not cell type specific. Inhibition of virus infection by the ␤-peptides occurred at the postattachment penetration step, with a 50% effective concentration of 3 M for the most-effective ␤-peptide. The ␤-peptides did not inactivate virions in solution, nor did they induce resistance to infection when cells were pretreated with the ␤-peptides. The ␤-peptides showed little if any toxicity toward Vero cells. These results raise the possibility that cationic ␤-peptides may be useful antiviral agents for HSV-1 and demonstrate the potential of ␤-peptides as novel antiviral drugs.Herpes simplex virus type 1 (HSV-1) is a significant human pathogen causing mucocutaneous lesions primarily in the oral mucosa (cold sores), as well as other sites. More-severe diseases caused by HSV-1 infection include encephalitis, meningitis, and blinding keratitis (65), and HSV-1 is the leading cause of blindness due to infection in developed countries (5). Following an initial infection, HSV-1 establishes latent infection of neurons in sensory ganglia of the host (29), from where it periodically reactivates and causes recurrent lesions at the site of primary infection. To date, none of the currently approved antivirals can eliminate an established latent infection. Because of the difficulties dealing with latency, preventing HSV-1 from entering the cell is an attractive antiviral strategy.HSV-1 entry is a complex process, involving multiple components on both the cell plasma membrane and the viral envelope. The initial interaction involves the binding of viral glycoprotein C (gC) or gB to cell surface heparan sulfate proteoglycan (58). Four viral glycoproteins, gB, gD, and the gH-gL heterodimer, are essential for the subsequent membrane fusion and entry steps (56,57). Following attachment, gD interacts with any of three cellular receptors: herpes virus entry mediator, nectin-1, and 3-O-sulfated heparan sulfate, leading to a conformational change in gD (12,13,21,22,34,39,54,62). This conformational change in gD is believed to trigger the formation of the fusion complex, which is thought to involve the sequential binding of gB to gD, followed by the binding of gH-gL to the gB-gD complex (10,20,23,24,36,41,50,62).