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Zika virus (ZIKV), isolated from macaques in Uganda in 1947, was not considered to be a dangerous human pathogen. However, this view has recently changed as ZIKV infections are now associated with serious pathological disorders including microcephaly and Guillain-Barre syndrome. Similar to other viruses in the Flaviviridae family, ZIKV expresses the serine protease NS3 which is responsible for viral protein processing and replication. Herein, we report the expression of an active NS3pro domain fused with the NS2B cofactor (NS2BLN NS3pro ) in a prokaryotic expression system and profile its specificity for synthesized FRET-type substrate libraries. Our findings pave way for screening potential intracellular substrates of NS3 and for developing specific inhibitors of this ZIKV protease.

Human papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human α-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data support a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid. IMPORTANCE Our study elucidates a new antiviral action for α-defensins against nonenveloped viruses in which HD5 directly interferes with a critical host-mediated viral processing step, furin cleavage of L2, at the cell surface. Blocking this key event has deleterious effects on the intracellular steps of virus infection. Thus, in addition to informing the antiviral mechanisms of α-defensins, our studies highlight the critical role of furin cleavage in HPV entry. Innate immune control, mediated in part by α-defensins expressed in the genital mucosa, may influence susceptibility to HPV infections that lead to cervical cancer. Moreover, understanding the mechanism of these natural antivirals may inform the design of therapeutics to limit HPV infection.

During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. IMPORTANCE Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

Once a virus infection is established, antiviral therapy is the option for the control of the viral infection. In principle, all the steps in the virus life cycle ranging from entry to release can be explored as molecular targets for antiviral therapy. In fact, the vast majority of antiviral drugs developed since the 1980s are anti-HIV drugs. These anti-HIV drugs block diverse steps of the HIV life cycle, including entry, reverse transcription, viral protein processing, and integration. Here, we will learn about a wave of newly identified drug targets and new drugs focusing on HBV, HCV, and influenza virus as well as HIV. In addition to small molecules, antibodies are used for antiviral therapy as well, under certain circumstances.

The major cellular tRNA(Lys) isoacceptors are tRNA(Lys1,2) and tRNA(Lys3). During the replication of human immunodeficiency virus 1 (HIV-1), tRNA(Lys3) is used to prime reverse transcription of the viral RNA genome into double-stranded DNA, which is then integrated into the host genome. The annealing of tRNA(Lys3) to 5'-terminal sequences of viral RNA is multi-staged, with an initial poor quality, cytoplasmic annealing promoted by the Gag precursor protein, followed by a more effective annealing imposed upon the Gag-annealed tRNA(Lys3) that occurs after viral protein processing, and that is facilitated by mature nucleocapsid (NCp7). The initial annealing by Gag is assisted by the architecture of an early viral assembly intermediate we term the "tRNA(Lys3) annealing complex" whose composition includes Gag, GagPol, viral RNA, lysyl-tRNA synthetase (LysRS), and the tRNA(Lys) isoacceptors. Our model proposes that the reverse transcriptase sequences in GagPol bind all tRNAs non-specifically, and that the cytoplasmic tRNA population to which GagPol is exposed is enriched in tRNA(Lys) isoacceptors due to a specific interaction between Gag and LysRS. We further predict a protein conformation within the annealing complex that not only promotes this tRNA(Lys) enrichment, but that also facilitates the transfer of tRNA(Lys3) from GagPol to the viral RNA where annealing is carried out by nucleocapsid sequences within Gag.

RNA helicase A (RHA) has been shown to promote HIV-1 replication at both the translation and reverse transcription stages. A prerequisite step for reverse transcription involves the annealing of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , the primer for reverse transcription, to HIV-1 RNA. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealing is a multistep process that is initially facilitated by Gag prior to viral protein processing. Herein, we report that RHA promotes this annealing through increasing both the quantity of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealed by Gag and the ability of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} to prime the initiation of reverse transcription. This improved annealing is the result of an altered viral RNA conformation produced by the coordinate action of Gag and RHA. Since RHA has been reported to promote the translation of unspliced viral RNA to Gag protein, our observations suggest that the conformational change in viral RNA induced by RHA and newly produced Gag may help facilitate the switch in viral RNA from a translational mode to one facilitating \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealing.

Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. Molecular studies of the virus became possible with the successful cloning of its genome in 1989. Although much work remains to be done regarding early and late stages of the HCV life cycle, significant progress has been made with respect to the molecular biology of HCV, especially the viral protein processing and the genome replication. This review summarizes our current understanding of genomic organization of HCV, features of the viral protein characteristics, and the viral life cycle.

A mathematical model for suppression of the hepatitis C virus RNA replicon replication in Huh-7 cell culture in the presence of potential drugs was built. There was a good agreement between the experimental and theoretical kinetic data for the decrease in the level of viral RNA in the cell in the presence of the competitive HCV NS3 protease inhibitor. Using the model, we verified the estimates for the efficiency of the effect of potential drugs on replication of viral RNA and viral protein processing. It was demonstrated that the tested drugs are most efficient at the replication step of viral RNA. The efficiency of the combined action of real and putative inhibitors target on the host and viral proteins was also studied. It was found that the action of the inhibitor at low concentrations on the host factors considerably enhances the suppressive effect on viral RNA replication in the presence of even the low affine NS3 protease inhibitors. The developed mathematical model may serve as a tool for the evaluation of the efficiency of potential drugs on the HCV genome.

Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to cytoplasmic, as input of exogenous DDX1 significantly altered both Rev sub-cellular localization from cytoplasmic to nuclear predominance and concomitantly increased HIV-1 viral production in these human astrocytes. We conclude that altered DDX1 expression in human astrocytes is, at least in part, responsible for the unfavorable cellular microenvironment for Rev function in these CNS-based cells. Thus, these data suggest a molecular mechanism(s) for restricted replication in astrocytes as a potential low-level site of residual HIV-1 in vivo.

Here we report a new chemical inhibitor against HIV-1 with a novel structure and mode of action. The inhibitor, designated as A1836, inhibited HIV-1 replication and virus production with a 50% inhibitory concentration (IC50) of 2.0 μM in an MT-4 cell-based and cytopathic protection antiviral assay, while its 50% cytotoxic concentration (CC50) was much higher than 50 μM. Examination of the effect of A1836 on in vitro HIV-1 reverse transcriptase (RT) and integrase showed that neither were molecular targets of A1836. The characterization and re-infection assay of the HIV-1 virions generated in the presence of A1836 showed that the synthesis of early RT products in the cells infected with the virions was inhibited dose-dependently, due in part to abnormal protein formation within the virions, thus resulting in an impaired infectivity. These results suggest that A1836 might be a novel candidate for the development of a new type of HIV-1 inhibitor. [BMB Reports 2015; 48(2): 121-126]

The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease. Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system. An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 μM for both Z-L-F-Q-CHN2and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2. The tripeptide Z-L-F-Q-CHN2blocked viral replication with an IC50value of 30 μM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 μM). Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide. These results indicate that DMK inhibitors of the 3C protease have antiviral potency. Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield.

The role of microtubules in the brefeldin A (BFA)-associated relocation of major histocompatibility complex (MHC) class II alphabeta chains (alphabeta) and the invariant chain (Ii) was characterized in Raji cells by the use of nocodazole (ND). BFA blocked the transport of alphabeta Ii proteins through the Golgi and redistributed them to the endoplasmic reticulum (ER) along with Golgi-resident enzymes. The result of the colocalization of processing enzymes and newly synthesized proteins was a downshift of alphabeta Ii molecular weight (MW) of 2 kDa, and their resistance to endoglycosidase H (endo H) after 6 hr of chase. ND by itself had no effect on the processing and transport of alphabeta to the cell surface. The addition of ND to BFA-treated cells downshifted alphabeta Ii by 4 kDa. Additionally, alphabeta Ii proteins remained sensitive to neuraminidase after 16 hr of chase. In vitro alpha-mannosidase treatment of immunoprecipitated alphabeta Ii generated a similar 4-kDa downshift of MW. Either 1-deoxymannojirimycin (DJN) or swainsonine (SWN) blocked the MW downshift caused by BFA + ND treatment. These observations indicated that in Raji cells, most of the BFA-associated relocations of cis-, medial Golgi proteins, and the addition of sialic acid from the trans-Golgi were microtubule-independent. The retrograde transport of the medial Golgi enzyme N-acetylglucosamine transferase, however, required microtubular function. Microtubule disrupters could affect BFA treatment of viral infections by further disrupting viral protein processing.

We have examined the effects of interferon (IFN)-α/β on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse–chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.

Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1–4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the cell-free infectivity of HIV-1 virus particles. Previous studies have demonstrated that vif increases viral infectivity at the time of virus production, most likely by affecting viral protein processing, virus assembly, or virus maturation. The effect of vif on the assembly and maturation of HIV-1 propagated in CEM, Jurkat, and SupT1 cells was examined by electron microscopy and goniometer analysis. CEM and Jurkat cells are nonpermissive and partially permissive for the replication of vif--defective viruses, respectively, while SupT1 cells are completely permissive. In CEM and Jurkat cultures, the morphology of immature vif+ and vif- virions was similar but immature virus particles were observed at a slightly higher frequency in cultures infected with the vif- virus. At later stages of virus maturation, however, nonhomogeneous packing of the core was detected in the majority of vif- virus particles produced in CEM and Jurkat cells. In the absence of vif, the cone-shaped virus core contained dense material in its broad end but, in contrast to vif+ virions, the material inside its narrow end appeared transparent. The narrow part of the vif- virus core was surrounded by a shell and was attached to the viral envelope by a core-envelope link structure. Vif- virus particles with a lateral body of core material adjacent to the viral envelope were also observed more frequently in CEM and Jurkat cultures. In contrast, in SupT1 cultures the morphology of mature vif+ and vif- virus particles was similar. These results suggest that vif is associated with an effect during the final stages of packing of the viral nucleoprotein core. This effect may be important for the infectivity of HIV-1 virus particles.

The West Nile virus strain Kunjin virus (WNV KUN ) NS4A protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, and evasion of host defense and is a major component of the WNV KUN RNA replication complex. We identified a highly conserved region ( 120 P-E-P-E 123 ) upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for WNV KUN replication. Single point mutations to alanine and a PEPE deletion mutation were created in a full-length infectious WNV KUN molecular clone. All mutations drastically impaired viral replication and virion production, except that of the P122A mutant, which was slightly attenuated. These mutations were subsequently transferred to a WNV KUN replicon to specifically assess effects on RNA replication alone. Again, all mutants, except P122A, showed severely reduced negative-sense RNA production as well as decreased viral protein production. Correspondingly, immunofluorescence analyses showed a lack of double-stranded RNA (dsRNA) labeling and a dispersed localization of the WNV KUN proteins, suggesting that replication complex formation was additionally impaired. Attempts to rescue replication via conservative mutants largely failed except for substitution of Asp at E121, suggesting that a negative charge at this residue is equally important. Analysis of viral protein processing suggested that cleavage of the 2K peptide from NS4A did not occur with the mutant constructs. These observations imply that the combined effects of proline and negatively charged residues within the PEPE peptide are essential to promote the cleavage of 2K from NS4A, which is a prerequisite for efficient WNV replication.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.

The glycosylation state of envelope glycoproteins in Human and Simian Immunodeficiency Viruses (HIV/SIV) is critical to viral infectivity and tropism, viral protein processing, and in virus evasion of the immune system. Using a rapid fluorescent two-dimensional gel based method coupled with enzymatic pre-treatment of virus with PNGase F (Peptide: N-Glycosidase F) and fluorescent 2D gels or 2D gel Western blotting, we show significant differences in the glycosylation patterns of two SIV strains widely used in animal models of HIV disease and vaccine studies. We also demonstrate the modification of a host protein important in HIV biology (HLA-DR) by O-GlcNAc. Further, this experimental pipeline allows for the identification of the modified protein and the site of N-linked glycosylation by fluorescent two-dimensional gel electrophoresis coupled with mass spectrometry (MS) and the qualitative and semi-quantitative assessment of viral glycosylation. The method is fully compatible with downstream glycomics analysis. This approach will permit correlation of virus glycosylation status with pathological severity and may serve as a rapid screen of viruses from physiological samples for further study by more advanced MS methodology.

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae , we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli , we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Background The structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol. Results In developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 × 106 TU/ml, unconcentrated). Conclusion Here, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (K(i) = 4 microM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC(50) values ranging from 7.6 to 84.5 microM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection ofor = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.

Human rhinoviruses are the major causative agents of the common cold. Because there are greater than 100 viral serotypes, little immunological protection is afforded to humans by prior rhinovirus exposure, which accounts for the high incidence of infection. In most cases, rhinovirus leads to a short self-limiting illness. However, for asthmatics, the elderly and immunocompromised patients, rhinovirus infection can lead to life-threatening complications. This has spurred a consistent effort over recent decades to identify effective treatments and preventions for rhinovirus infection. While some work has focused on alleviating the symptoms induced as a result of inflammatory pathways stimulated by rhinoviruses, the majority of the research has been focused on limiting or preventing viral infection altogether. Various approaches have been taken to halt rhinovirus infection. Prevention of virus-cell interaction has been the aim of research on viral capsid binders and cell receptor blockers. Interference with correct viral protein processing is the goal of the design and testing of protease inhibitors. Current work is attempting to interfere with viral RNA replication by testing silencing RNA molecules. In this review, we will discuss recent advances in the development and testing of human rhinovirus therapeutics.

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.

Limited proteolysis of most large protein precursors is carried out in vivo by the subtilisin-like pro-protein convertases. Many important biological processes such as peptide hormone synthesis, viral protein processing and receptor maturation involve proteolytic processing by these enzymes, making them potential targets for the development of novel therapeutic agents. However, the efficient development of such molecules requires a better understanding of the molecular mechanisms of proteolytic protein processing. Herein, we review the most recent findings on the molecular aspects of subtilisin-like convertase activity, such as the structural analysis of the proteases, the mechanisms of enzyme/substrate specificity, their interaction with other proteins such as 7B2, and the comparative tissue and cellular distribution of the enzymes and their substrates. These data are then used as a background for the review of the known biological functions of subtilisin-like pro-protein convertases, the reported clinical cases involving proteolytic processing defects and, finally, the ongoing development of new therapeutic inhibitor molecules based on this knowledge.

A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.

The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 × 106, having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3′ and 5′ termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence (“positive” polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3′-G2-G1–5′. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed.

Zinc ion inhibits the posttranslational cleavages of human rhinovirus-1A, encephalomyocarditis virus, and poliovirus polypeptides. Each virus displayed a different susceptibility to zinc. However, in each case the cleavages of the capsid precursor and the cleavages analogous to the C → D → E conversion in encephalomyocarditis virus were most sensitive to zinc. Higher concentrations of zinc resulted in the buildup of even larger precursor polypeptides of a size between 106,000 and 214,000 daltons. The sizes of these polypeptides and the relative position of their gene loci on the viral RNA were determined. These data were used to place these polypeptides in the over-all scheme of viral protein processing.

A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0–VP3). The data indicated that processing of the polyprotein P l by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.

Serum samples from 27 patients infected with human T-cell lymphotropic virus type III (14 with acquired immune deficiency syndrome [AIDS] and 13 with AIDS-related complex) were examined for antibodies to viral proteins by the Western blot method and with four different commercial solid-phase enzyme-linked immunosorbent assays (ELISAs). Virus-specific bands on blots at molecular masses of 64, 55, 53, 41, 31, 24, and 17 kilodaltons were observed. Rank correlation matrices were calculated to relate the intensity of viral bands, stage of illness, and ELISA kit optical densities (ODs). Groups of bands tended to covary in intensity: p17, p24, and p55 (gag gene products); p53 and p64 (pol gene products); and p31 (pol/endonuclease gene product) and p41 (env gene product). Blots of sera from AIDS-related complex patients usually showed strong activity against all viral proteins, while those of sera from AIDS patients characteristically showed strong reactivity only at the pol/endonuclease and env bands. For one ELISA kit (Abbott Laboratories, North Chicago, Ill.), ODs correlated well with the env and pol band intensity scores, while ELISA ODs with other kits (from Litton Industries, Sunnyvale, Calif.; Electro-Nucleonics, Inc., Fairfield, N.J.; and E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.) correlated closely with gag band intensity scores. We conclude that human T-cell lymphotropic virus type III Western blot patterns are determined by (i) viral protein processing pathways and (ii) the stage of illness of the patient and may reflect (iii) the ELISA method used for serum screening.