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PubMed1991

HIV-1 proteinase is required for synthesis of pro-viral DNA.

HIV-1 proteinase activity is thought to occur primarily post-integration by cleaving the viral Gag and Gag-Pol polyproteins. Its role in the pre-integration stages of viral replication, however, has not been studied in detail. Here we report that a synthetic peptide analogue, UK-88,947, which is a specific inhibitor of purified HIV-1 proteinase, inhibits the processing of the viral polyproteins in cultures of HIV-1 infected cells and prevents the formation of mature, infectious virions. Analysis of DNA from HIV-1 infected cells treated with UK-88,947 showed that viral DNA synthesis was inhibited when the compound was added to cultures one hour before infection. Similar results were obtained when AZT was used. Neither HIV-1 reverse transcriptase or the replication of FIV are inhibited by UK-88,947.

Amino Acid Sequence↗

PubMed1994

Phospholipid prodrug inhibitors of the HIV protease. Antiviral activity and pharmacokinetics in rats.

The aspartyl protease of the human immunodeficiency virus (HIV) is an important target for chemotherapeutic intervention because of its key role in cleaving the HIV gag-pol polyprotein during viral assembly and budding. Short peptides and peptidomimetics, which bind to the active site of the HIV aspartyl protease and inhibit processing of the polyprotein, have been synthesized. These compounds are active against HIV in vitro, but many face substantial development problems because of their rapid elimination from the body in bile and urine. Refinement of these agents appears to be necessary if they are to become useful clinically. Recently, we developed a novel chemical strategy for increasing plasma levels of HIV protease inhibitory peptides, which involves the attachment of a biodegradable phospholipid group to the C-terminus of a pentapeptide, iBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194). We coupled phosphatidylethanolamine to the C-terminal valine of 7194 to make a phospholipid prodrug (7196). In vitro assays in HT4-6C cells infected with HIV-1 showed that the antiviral activity of the C-terminal phospholipid prodrug, 7196, was equal to that of the free peptide, 7194. Similar results were obtained in vitro when a related pentapeptide (7140) was derivatized at the N-terminal with dipalmitoylphosphatidylethanolamine-succinic acid (7172). Tritium-labeled 7194 and 7196 were prepared and injected intravenously into rats at 3 mumol/kg; then the plasma was assayed for native compound and metabolites by HPLC radioactivity flow detection. The peak plasma level of the tritium-labeled lipid prodrug (7196) was 36 microM versus 1.6 microM for the free protease inhibitor pentapeptide (7194). The area under the curve of the phospholipid prodrug (7196) was 48-fold greater and its mean residence time was increased 43-fold versus the free peptide (7194). Phospholipid prodrugs appear to offer an alternative approach to optimizing in vivo performance of HIV protease inhibitors and other small peptides.

Amino Acid Sequence↗

PubMed1988

Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag).

Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins. Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action. In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag. We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E. coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing.

Amino Acid Sequence↗

PubMed1991

Molecular structure of the Japanese hepatitis C viral genome.

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524-9528) indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase, NTPase and RNA-dependent RNA polymerase.

Amino Acid Sequence↗

PubMed1991

Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli.

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.

Animals↗

PubMed1991

Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses.

A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomes of several diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protease of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal p28 protein from the polyprotein. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteases was obtained, leading to tentative identification of the papain-like protease as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouping between the sequences of the proteases of alpha-viruses, and poty- and bymoviruses was revealed. It was noted that papain-like proteases of positive-stranded RNA viruses are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same virus class. A novel conserved domain of unknown function has also been identified which flanks the papain-like proteases of alpha-, rubi- and coronaviruses.

Alphavirus↗

PubMed1983

Feline sarcoma virus-specific transformation-related proteins and protein kinase activity in tumor cells.

Polyproteins (gag-fes) encoded by the Synder-Theilen (ST) and the Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) were previously shown to be associated with mink or rat cells that were nonproductively transformed in vitro. In the present study we demonstrated that the same gag-fes proteins were found in cat cells transformed in vitro. Of greater importance, these transformation-related proteins were also in cells taken from fresh biopsies of FeSV-induced tumors. Cells from fibrosarcomas induced with ST-FeSV had gag-fes proteins that were characteristic of this strain. Fibrosarcomas and melanomas were induced with GA-FeSV and both types of tumors contained the protein that is characteristic of cells transformed in vitro with this virus. Expression of these proteins in cultured tumor cells appeared to be independent of the passage level. Based on two-dimensional tryptic peptide analysis, the gag-fes proteins of cat tumor cells appeared to be indistinguishable from those found in cells transformed in vitro. The polyproteins of the cat tumor cells have a closely associated protein kinase activity, as demonstrated in the in vitro assay, and phosphorylated tyrosine residues. Gag-fes proteins of either the ST or GA class were not present in cell cultures initiated from five spontaneous cat tumors.

Animals↗

PubMed1983

3Y1 rat cells are defective in processing of the envelope precursor protein of AKR virus.

Rat 3Y1 cells were infected by AKR virus through microinjection of molecularly cloned proviral DNA. Based on a strong immunofluorescence using anti-p30 as an antiserum a cell clone (RESA-2) was selected that had a high expression of viral antigens. Subsequent restriction analysis of its DNA revealed that the RESA-2 clone contained at least 30 apparently intact integrated proviruses per genome. There was an apparently normal synthesis and processing of gag and pol gene products. The viral envelope precursor polyprotein gPr82env, however, did not yield the major envelope glycoprotein gp70. The gag precursor polyprotein, Pr65gag, as well as the gPr82env from RESA-2 cells were identified as AKR viral proteins by gel electrophoresis of hydroxylamine cleavage fragments. The virions formed by RESA-2 cells lacked gp70 and were noninfectious. After fusion of RESA-2 cells and mouse cells an infectious N-tropic virus was produced. The results indicate that rat 3Y1 cells lack (a) factor(s) necessary for the correct processing of gPr82env. The high incidence of abortive infections of murine leukemia virus (MLV) in susceptible rat cells reported by others is therefore probably due to defective particles in the virus stock and/or to the lack of (a) cellular factor(s) necessary for reverse transcription and subsequent integration of the viral genome.

AKR murine leukemia virus↗

PubMed1983

Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor.

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.

Animals↗

PubMed1984

Isolation of a new feline sarcoma virus (HZ1-FeSV): biochemical and immunological characterization of its translation product.

A new strain of feline sarcoma virus, designated HZ1-FeSV, was isolated from a 4-year-old domestic cat with multicentric fibrosarcoma. A primary tumor cell line was established and virus produced from that line was found to induce foci in feline embryonic lung fibroblasts (FLF3) and mink lung fibroblasts (CCL64) in tissue culture and fibrosarcomas in inoculated 10-week-old kittens. The derivation of transformed nonproducer clones of FLF3 and CCL64 cells containing helper virus-rescuable, focus-forming activity indicated that HZ1-FeSV was defective for replication. The only discernible translation product of the HZ1-FeSV genome in cultured cells was a 100,000-Da polyprotein (P100) which contained amino-terminal sequences of the FeLV gag gene precursor protein covalently linked to a sarcoma virus-specific domain. Immunoprecipitates containing P100 exhibited a protein kinase activity capable of phosphorylating tyrosine residues of P100. Immunologically, P100 was highly cross-reactive with gag-fes polyproteins encoded by two previously characterized strains of FeSV, the GA- and the ST-FeSV. By comparison of methionine-containing tryptic peptides, the HZ1-FeSV protein was shown to be more closely related to the GA-FeSV protein than to the ST-FeSV protein, but to be distinguishable from both other proteins.

Animals↗

PubMed1985

The genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies.

In mRNA-dependent extracts of Krebs-2 cells, RNAs from attenuated strains of poliovirus type 1 and type 3 exhibited diminished template activity as compared to RNAs from the respective virulent counterparts. This defect appeared to be due to the impaired initiation of viral polyprotein synthesis as evidenced by a relatively low level of accumulation of polypeptide 1a (which corresponds to an NH2-terminal region of the polyprotein) in samples programmed with RNAs from attenuated strains. In reticulocyte lysates, where poliovirus RNA is translated predominantly from abnormal (internal) sites [Dorner et al. (1984) J. Virol. 50, 507-514], this difference in the overall template activity of the attenuated and virulent poliovirus genomes was less pronounced, but the correct initiation (as judged by polypeptide 1a accumulation) was again more efficient on RNAs from virulent strains. It is suggested that template deficiency is a factor contributing to the attenuated phenotype of poliovirus strains studied. A possible involvement of nucleotide sequences located far upstream from the initiator codon in the control of translation of poliovirus genome is briefly discussed.

Cell Line↗

PubMed1986

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.

Amino Acid Sequence↗

PubMed1986

The effects of 2-deoxyglucose and tunicamycin on the biosynthesis of the murine mammary tumor virus proteins, and on the assembly and release of the virus.

The role of glycosylation in the biosynthesis, processing, and shedding of the murine mammary tumor virus (MuMTV) glycoproteins and in virus production was investigated in a clonal mammary tumor cell line, GR-3A, using two inhibitors of protein glycosylation, 2-deoxyglucose (2-DG) and tunicamycin (TM). It was found that both 2-DG and TM completely inhibited the synthesis of the MuMTV envelope precursor polyprotein, Pr70env, and, as a consequence, the synthesis of the viral glycoproteins gp52 and gp36. By contrast, the synthesis of Pr73gag, the polyprotein precursor of the internal structural proteins of the virus, was only inhibited by 10-15% by 2-DG and TM. Although 2-DG and TM blocked the synthesis of Pr70env, a new polypeptide, related to gp52 and gp36, with a mol wt of 60,000 (P60env) was found to be synthesized in the treated cells. The P60env molecules appeared to be degraded intracellularly since they were not found to (1) undergo site-specific cleavage; (2) accumulate inside the cell or on the cell surface; (3) be secreted into the culture medium; and (4) be incorporated into the virions produced during the drug treatment. In spite of the lack of gp52 and gp36 synthesis in the presence of TM and 2-DG, mature MuMTV particles containing the characteristic surface projections known to be composed of gp52 and gp36 continued to be assembled and released at a reduced rate for at least 30 hr. In addition, the buoyant density and the polypeptide composition of the particles were found to be identical to virions produced by untreated cells. Thus, the virions assembled and released during 2-DG and TM treatment were not defective. Our investigations into the origin of gp52 and gp36 in these particles revealed that both molecules were synthesized prior to 2-DG and TM treatment and continued to be incorporated, along with the newly synthesized viral core proteins, into budding virions during the drug treatment. Furthermore, we found that gp52 and P75env (an aberrant form of Pr70env) that were not incorporated into virions continued to be shed normally from the cell during drug treatment. In conclusion, our results suggest that MuMTV assembly is not dependent on the synchronized synthesis of the viral core and envelope polypeptides, and that the assembled virions contain the correct ratio of these polypeptides, even when their ratio in the cell varies.

Animals↗

PubMed1987

Complete nucleotide sequence of the genome of coxsackievirus B1.

The complete nucleotide sequence of the genome of the coxsackievirus B1, a human enterovirus that belongs to the Picornaviridae, was determined by using molecular cloning and rapid sequence analysis techniques. Sequence analysis of the cloned cDNAs revealed that the virion RNA was 7389 nucleotides long and polyadenylylated at the 3' terminus. Similar to other picornavirus genomes, a single large open reading frame was identified. The translated sequence starts at nucleotide position 742 and ends at 7287 of the genome. Thus, the viral polyprotein should consist of 2182 amino acids. When the predicted amino acid sequence of the viral polyprotein was compared with those of other human enteroviruses such as polioviruses, a striking sequence homology was observed, especially in viral proteins 1B, 2C, and 3D. This allowed us to predict precise map locations of the viral structural and nonstructural proteins on the genome, although two proteolytic processing sites, between 1D and 2A and between 2B and 2C, were obscure. The result presented here implied important information with respect to the genetical variation of human enteroviruses.

Amino Acid Sequence↗

PubMed1987

The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins.

We recently cloned a full-length DNA copy of the dengue type 4 virus genome. Analysis of the 5' terminal nucleotide sequence suggested that the three-virion structural proteins are synthesized by proteolytic cleavage of a polyprotein precursor which is encoded in one open reading frame. We now present the remaining sequence of the dengue type 4 virus genome which codes for the nonstructural proteins. The entire genome, which is 10,644 nucleotides in length, contains one long open reading frame which codes for a single large polyprotein 3386 amino acids in length. Alignment of the dengue nonstructural protein sequence with that of other flaviviruses, including yellow fever and West Nile viruses, revealed that significant homology exists throughout the entire nonstructural region of the dengue genome and this allowed tentative assignment of individual nonstructural proteins in the following order: NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5-COOH. Processing of the nonstructural proteins appears to involve two types of proteolytic cleavage: the first occurs after a long hydrophobic signal sequence and the second occurs at a junction between two basic amino acids and a small polar amino acid. A notable exception is the cleavage at the N-terminus of the dengue NS3 which may take place at the junction between Gln-Arg and Ser. Comparative analysis suggests that dengue NS3 and NS5 may be involved in enzymatic activities related to viral replication and/or transcription. Putative nonstructural proteins NS2a, NS2b, NS4a, and NS4b are extremely hydrophobic, suggesting that these proteins are most likely associated with cellular membranes.

Amino Acid Sequence↗

PubMed1988

Insights into Theiler's virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains.

Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which can be divided into two subgroups based primarily on their neurovirulence after intracerebral inoculation: the highly virulent GDVII group and the less virulent TO strains. To begin to elucidate the molecular basis of neurovirulence of the two TMEV subgroups, we have cloned and sequenced the entire 8105 nucleotide RNA genome of the highly virulent GDVII virus and compared it to the less virulent BeAn 8386 virus (D. C. Pevear, M. Calenoff, E. Rozhon, and H. L. Lipton (1987) J. Virol. 61, 1507-1516). The viruses are 90.4% identical at the nucleotide level. The highest level of nucleotide identity is in the 5' and 3' noncoding regions of the RNAs (95.5 and 99.2%, respectively): regions believed to be important for control of viral RNA synthesis, initiation of translation, encapsidation, and virion uncoating. The 2303 amino acid polyproteins of BeAn and GDVII viruses are 95.7% identical at the amino acid level (99 of 2303 residues differed). Thirty-nine of these amino acid differences occur in the three surface coat proteins, VP1 (20 differences), VP2 (10 differences), and VP3 (9 differences), while the remainder of the changes are distributed throughout the polyprotein. Although these levels of identity are too low to determine where neurovirulence maps based solely on nucleotide sequence analysis, having the complete sequence will facilitate construction of recombinant BeAn-GDVII viruses to be used for this purpose.

Amino Acid Sequence↗

PubMed1989

Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells.

To study the capability of Spodoptera frugiperda (fall armyworm; Sf9) cells to synthesize and process mature rubella virus (RV) proteins, a cDNA encoding the structural envelope glycoproteins, E1 (58 kDa) and E2 (42-47 kDa) were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during infection under the transcriptional regulation of the polyhedrin gene promoter. By immunoblot analysis with antibodies directed against purified RV, the individual proteins E1 and E2, and human convalescent serum, a polyprotein precursor migrating with an apparent molecular weight of 90-95 kDa was identified in Sf9 cells infected with the recombinant baculovirus, Ac701-RVE. In addition, two proteins migrating somewhat faster than authentic viral E1 and E2 were resolved. Pulse-chase labeling experiments in the absence and presence of tunicamycin, as well as treatment of the recombinant proteins with endo-beta-N-acetyl-D-glucosaminidase H indicated that the recombinant proteins are glycosylated and that the E1 and E2 apoproteins, respectively, were similar in size as compared to their in vitro synthesized counterparts. The recombinant protein products were further detected by some monoclonal antibodies directed against RV. The results presented here indicate that a polyprotein containing the envelope glycoproteins of RV is expressed and proteolytically cleaved in lepidopteran insect cells to form two proteins which resemble authentic E1 and E2. The baculovirus may therefore be suitable for the abundant expression of RV antigen.

Animals↗

PubMed1989

Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase.

The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.

Aprotinin↗