Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide proton exchange rates for individual glycoforms of RNase B and unglycosylated RNase A showed that while the 3D structure was unaffected, glycosylation decreased dynamic fluctuations throughout the molecule. There was individual variation in the NH-ND exchange rates of the same protons in different glycoforms, demonstrating the effects of variable glycosylation on dynamic stability. Consistent with the overall decrease in flexibility, and with the possibility that all of the sugars may afford steric protection to susceptible sites, was the finding that each of the glycoforms tested showed increased resistance to Pronase compared with the unglycosylated protein. In a novel sensitive assay using double-stranded RNA substrate, the different glycoforms showed nearly a 4-fold variation in functional activity; molecular modeling suggested that steric factors may also play a role in modulating this interaction.

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of ϳ64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/ LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3-5 exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drugresistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

Human papillomaviruses (HPV) are associated with benign lesions and show specificity towards the location or tissues that they infect. HPVs are responsible for warts. Among more than 60 different HPV types known to occur in humans, a strong association has been found between types 16 and 18 and cervical cancer, and such an association is also suspected for types 31, 33, 35, 45, 51, 52, and 56. We describe the effects of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC), following local intratumoral injection, in a 69-year-old woman with hypopharyngeal and esophageal papillomatous lesions, polymerase chain reaction (PCR) positive for HPV types 16 and 18, that relapsed after surgery and that also failed to respond to Nd-Yag laser photocoagulation and alpha-interferon treatment (6 x 10(6) U five times a week for 4 weeks followed by three times a week for 2 months). HPMPC was given at 1.25 mg/kg, with a sclerosing needle, through the biopsy channel of a video-endoscope, directly into the tumor, from March until July 1993 at seven different occasions. The first four injections were given at an interval of 1 week at the level of the hypopharynx. The next three injections were given at an interval of 3 to 5 weeks. During the fourth to the seventh session, half of the dose was injected into the hypopharyngeal and the other half into the esophageal tumor. Three further injections of HPMPC were administered at the level of the esophageal tumor in September 1993 with 2-week intervals. After HPMPC treatment, the lesions became smaller and flat until they completely disappeared.(ABSTRACT TRUNCATED AT 250 WORDS)

The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a ϳ15-foldincreased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

Drug-resistant strains of herpes simplex virus type 1 (HSV-1) were selected under the pressure of (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC, cidofovir) and adenine (HPMPA) and 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA, adefovir) and 2,6-diaminopurine (PMEDAP). HPMPC-resistant (HPMPC r ) and HPMPA r strains were cross-resistant to one another, but they remained sensitive to foscarnet (PFA), acyclovir (ACV) and the PME derivatives, while the PMEA r and PMEDAP r strains showed cross-resistance to PFA and ACV. The PMEA

Gelatinase B is a regulated matrix metalloproteinase with an important role in the remodelling of extracellular matrices and of basement membranes. To study the structure and function of gelatinase B in the mouse, the cDNA was cloned from a macrophage cell line (WEHI-3). Using this cDNA, a cosmid clone with the mouse gene was isolated. The complete gene (8 kbp) was sequenced and compared with the human gene structure. There was 78% similarity at the cDNA level and the exodintron structure of the murine gene was similar to the human counterpart. At the 5' untranslated side, 1200 bp of the promoter/enhancer region were sequenced and found to contain several transacting-factor-binding sites. The mRNA transcription-initiation site was determined by non-isotopic primer-extension analysis. Polymerase-chain-reaction amplification of cDNAs yielded indirect evidence for a reverse-transcription stop in WEHI-3 cell mRNA. The DNA-derived mouse-protein structure exhibited 82% similarity with the human one. This similarity was functionally reflected by cross-reactivity of the mouse protein with an antiserum against human gelatinase B. The production of murine gelatinase B was studied at the protein level by zymography and at the mRNA level by Northern blot analysis. In WEHI-3 cells the gelatinase B protein is induced by bacterial lipopolysaccharide, phorbol ester, double-stranded RNA and the cytokine interleukin-1 . Regulation of activity and structural heterogeneity of gelatinase B in WEHI-3 cells were shown to occur at the gene regulatory level, by expression of the matrix metalloproteinase inhibitor TIMP-1, and by glycosylation of the secreted protein.Matrix metalloproteinases (MMP) belong to a class of proteolytic enzymes which act together in degrading most components of extracellular matrices and of basement membranes. Physiological processes in which MMP play a role include embryonic growth and development, migration of blood leukocytes into tissues and tissue remodelling (Matrisian, 1992). Elevated levels of certain MMP are believed to be associated with various pathological states such as tumorcell invasion and metastasis and inflammatory processes such as rheumatoid arthritis and multiple sclerosis (Opdenakker and Van Damme, 1992a,b Note. The novel nucleotide sequence data published here have been deposited with the EMBL GenBank and DDBT sequence data banks and are available under the accession numbers X72794 and X72795, for the gene and cDNA, respectively.

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