Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor. hCMV protease is a serine protease, although it has little homology to other clans of serine proteases. Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.

Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5-and 2.4-Å resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.Human papillomaviruses (HPVs) 1 are the etiological agents of malignant and benign lesions of the differentiating squamous or mucosal epithelium, notably of cervical cancer. Approximately 25 HPV types replicate in mucosal tissues of the anogenital tract. HPV16, -18, and -31 are the most prevalent "high-risk" types found in pre-cancerous or malignant lesions of the cervix. HPV6 and -11 are the most common "low-risk" types, which cause benign genital warts (condyloma acuminata), a less serious condition but one of the most common sexually transmitted diseases (1). Currently, no specific antivirals are available for the treatment of HPV infections.The small circular double-stranded DNA genome of papillomavirus is actively maintained as a multicopy episome in the nucleus of infected epithelial cells. This process is dependent on replication of the viral genome by the viral E1 and E2 proteins, in conjunction with the host DNA replication machinery. E2 is a sequence-specific DNA-binding protein that has a number of functions in the viral lifecycle. In addition to its role in the initiation of viral DNA replication, E2 is involved in regulating the transcription of viral genes (2-7), and in the segregation of the viral genome during cell division (8, 9). As a replication initiation factor, E2 binds with high affinity to specific sites located within the viral origin (ori) to help recruit it to the E1 helicase (10 -13). Formation of a ternary complex between E1, E2, and the origin relies not only on the interaction of E1 and E2 with specific DNA sequences at the origin but is also critically dependent on a direct interaction between these two proteins (14 -18).The 40-kDa E2 prote...

The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.T he hepatitis C virus (HCV)-encoded NS3 protease is essential for viral replication and has long been considered an attractive target in drug design efforts (3, 5). NS3 protease inhibitors (PIs) can induce substantial reductions in HCV RNA plasma levels, and several candidates have progressed through clinical development to offer improved treatment options (for a review, see reference 27). Two PIs, boceprevir and telaprevir, were recently approved for use in combination with pegylated interferon (Peg-IFN) and ribavirin (1,6,7,19). The selection of drug-resistant variants is commonly observed in patients experiencing virologic rebound during treatment with PIs (16,[20][21][22]24).BI 201335 is a potent HCV NS3/4A PI (15, 28) currently in phase 3 clinical trials in combination with Peg-IFN and ribavirin as well as phase 2 assessment with other HCV direct acting antivirals in IFN-sparing regimens. BI 201335 exhibited a profound reduction in viral load when administered for 14 days as monotherapy in treatment-naïve patients or for 28 days in combination with Peg-IFN and ribavirin in treatment-experienced patients (16). In these studies, viral breakthrough was observed in most patients on monotherapy, whereas breakthrough was less frequent in patients undergoing combination treatment. Distinct resistant NS3 variants R155K and D168V predominated for genotype 1a and 1b (GT 1a and GT 1b), respectively (8,16).This study was designed to evaluate the genotypic and phenotypic profiles of the resistant variants that emerged during in vitro selection in the presence of BI 201335 in the replicon system and to relate these results to clinical observations. Replicons resistant to BI 201335 were selected in GT 1a H77 and GT 1b CON-1 replicon cell lines in the presence of 2 concentrations (100ϫ and 1,000ϫ drug concentration required to reduce HCV RNA or the luciferase reporter levels by 50% [EC 50 ]) of drug for 3 weeks and G-418 as previously described (9). With the lower concentration of BI 201335, resistant variants encoding NS3 changes at residues 155, 156, and 168 were selected with the GT 1b replicon, with D168G as the predominant variant (55%). R155K was the predominant variant (68%) selected with the GT 1a replicon (Table 1) and is consistent with the predominant variant selected in GT 1a HCV-infected patients (16). At the higher concentration of BI 201335, essentially only D168 variants were selected with D168 A and V as the predominant variants in both genotypes.In order to confirm that the mutations ob...