A cross-shaped terahertz metamaterial absorber for brain cancer detection.

The article presents, for the first time, a terahertz metamaterial absorber (TMA) designed in the shape of a cross consisting of four orthogonally positioned horn-shaped patches in succession, to detect brain cancer cells. The design exhibits the property of mu-negative material, indicating magnetic resonance. The proposed TMA has achieved an impressive absorption rate of 99.43% at 2.334 THz and a high Q-factor of 47.15. The sensing capability has been investigated by altering the refractive index of the surrounding medium in the range of 1.3 to 1.48, resulting in a sensitivity of 0.502 THz/RIU. The proposed TMA exhibits complete polarization insensitivity, highlighting this as one of its advantageous features. The adequate sensing capability of the proposed TMA in differentiating normal and cancerous brain cells makes it a viable candidate for an early and efficient brain cancer detector. This research can be the foundation for future research on using THz radiation for brain cancer detection.

Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

Gene expression of nonsegmented negative strand RNA viruses.

Nonsegmented negative strand RNA viruses comprise major human and animal pathogens in nature. This class of viruses is ubiquitous and infects vertebrates, invertebrates, and plants. Our laboratory has been working on the gene expression of two prototype nonsegmented negative strand RNA viruses, vesicular stomatitis virus (a rhabdovirus) and human parainfluenza virus 3 (a paramyxovirus). An RNA-dependent RNA polymerase (L and P protein) is packaged within the virion which faithfully copies the genome RNA in vitro and in vivo; this enzyme complex, in association with the nucleocapsid protein (N), is also involved in the replication process. In this review, we have presented up-to-date information of the structure and function of the RNA polymerases of these two viruses, the mechanisms of transcription and replication, and the role of host proteins in the life-cycle of the viruses. These detailed studies have led us to a better understanding of the roles of viral and cellular proteins in the viral gene expression.

Involvement of actin microfilaments in the transcription/replication of human parainfluenza virus type 3: possible role of actin in other viruses.

Multifunctional involvement of actin microfilaments during viral infection has been documented in many studies. The molecular mechanism underlying this important host-virus interaction, however, remains poorly understood. We have investigated the role of actin microfilaments in the life cycle of human parainfluenza virus type 3 (HPIV3), a paramyxovirus that causes severe respiratory illness in children. In vitro transcription with purified viral ribonucleoprotein (RNP) complex showed a requirement of cellular actin, in the polymeric form, for mRNA synthesis in vitro. This was further confirmed by using recombinant actin, which interacted with the viral RNP and also activated mRNA synthesis in vitro. Consistent with the role of the polymeric form of actin, the actin microfilaments of the cytoskeletal framework participate in the virus replication in vivo. Biochemical and immunological analyses revealed the association of viral RNPs with cytoskeletal framework during early stages of infection, and involvement of these RNPs in the synthesis of mRNAs and genome-length RNA. Immunofluorescent labeling and confocal microscopy showed that the viral nucleocapsids colocalize with the actin microfilaments. Treatment of cells with cytochalasin D, which depolymerizes actin microfilaments, inhibited viral RNA synthesis and RNP accumulation. These data indicate that actin microfilaments play a critical role in HPIV3 life cycle, specifically at the level of viral transcription and replication. Involvement of the cytoskeletal framework in the life cycle of several viruses containing RNA and DNA genomes is reviewed.

Human parainfluenza virus type 3 upregulates ICAM-1 (CD54) expression in a cytokine-independent manner.

Human parainfluenza virus type 3 (HPIV3) causes bronchiolitis, pneumonia, and croup in newborns and infants. Several studies have implicated intercellular adhesion molecule-1 (ICAM-1) in inflammation during infection by viruses. In this study, we investigated the potential for HPIV3 to induce ICAM-1 in HT1080 cells. FACS analysis showed that HPIV3 strongly induced ICAM-1 expression in these cells. The ICAM-1 induction was significantly reduced when the virions were UV inactivated prior to infection, indicating that ICAM-1 induction was mostly viral replication dependent. Culture supernatant of HPIV3-infected cells induced ICAM-1 at an extremely low level, indicating that virus-induced cytokines played only a minor role in the induction process. Consistent with this, potent inducers of ICAM-1 such as IFN-gamma, TGF-beta, and TNF-alpha were absent in the culture supernatant, but a significant amount of IFN type 1 was present. By using U2A cells, which are defective in IFN type I signaling, we confirmed that ICAM-1 induction by HPIV3 occurred in a JAK/STAT signaling-independent manner. These data strongly indicate that HPIV3 induces ICAM-1 directly by viral antigens in a cytokine-independent manner; this induction may play a role in the inflammation during HPIV3 infection.

Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro.

The L and NS proteins of vesicular stomatitis virus were purified from transcribing ribonucleoprotein complex and were used to study their requirements and functions during reconstitution of RNA synthesis in vitro. The requirements for L and NS proteins for optimal RNA synthesis were found to be catalytic and stoichiometric, respectively. Addition of increasing amounts of NS protein to N-RNA template and saturating L protein, the ratio of N-mRNA to leader RNA synthesis increased linearly. In contrast, when the concentration of L protein was increased the corresponding ratio remained constant. These results, coupled with the observation that the L protein is involved in the initiation of RNA synthesis, suggest that the NS protein is involved in the RNA chain elongation step. The NS protein possibly interacts with both the L protein and the template N-RNA and unwinds the latter to facilitate the movement of L protein on the template RNA.

Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus.

A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.

Specific binding of guanosine 5'-diphosphate with the NS protein of vesicular stomatitis virus.

A soluble protein fraction containing L, NS, G and M proteins of vesicular stomatitis virus was prepared by treatment of Triton-disrupted virions with 0.8M NaCl. Incubation of the soluble fraction with beta-32P GDP followed by analysis of the proteins by polyacrylamide gel electrophoresis showed specific labeling of the NS protein. The NS-GDP complex was sensitive to phosphatase treatment, suggesting non-covalent binding. No binding of GDP to NS protein was detected when the soluble fraction was pre-heated at 100 degrees C for 1 min. or Mg++ was omitted from the incubation mixture. The binding was inhibited by ATP consistent with competition for a common nucleotide binding site.

Specific interactions of vesicular stomatitis virus L and NS proteins with heterologous genome ribonucleoprotein template lead to mRNA synthesis in vitro.

Two dissociable proteins, L and NS, and N-RNA template were purified from two serologically distinct vesicular stomatitis viruses, Indiana [VSV(IND)] and New Jersey [VSV(NJ)]. Requirements for RNA synthesis in heterologous reconstitution reactions in vitro were studied. The L and NS proteins of VSV(NJ) failed to synthesize full-length leader RNA and mRNAs in vitro when reconstituted with N-RNA(IND) template. However, when purified homologous NS(IND) was added to the reaction mixture, mRNA synthesis ensued. The requirements for transcription of N-RNA(NJ) template were different from those for N-RNA(IND). For RNA synthesis, transcription specifically required L(NJ), but the NS(NJ) and NS(IND) proteins were interchangeable. This suggests that there are specific domains on the L(NJ) protein, at which NS proteins of both serotypes may interact to form an active RNA polymerase complex, whereas L(IND) lacked such domains for interaction with NS(NJ). The function of the L protein appeared primarily to initiate RNA chains, and the NS protein was required for chain elongation. The results of these in vitro complementation experiments are discussed in light of previous in vivo complementation studies.

Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.

A simple system that allows expression and packaging of a foreign gene by human parainfluenza virus type 3 (HPIV-3) has been described. First, a cDNA was constructed to encode an internally deleted version of HPIV-3 genome RNA. The viral genes were replaced with a negative sense copy of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In vitro run-off transcription with T7 RNA polymerase synthesized an 870 nucleotide RNA that contained the antisense coding region of the CAT gene flanked by the transcription regulatory sequences and the 3' and 5' end extracistronic sequences of the HPIV-3 genome. When introduced into cells that are infected with HPIV-3, this RNA was amplified and the reporter gene was expressed, as measured by the CAT activity in the cell extract. Furthermore, the synthetic RNA was packaged into infectious virions. The addition of two extra nucleotides at the 5' end of the parental trailer region decreased the CAT activity by more than 90%, suggesting a requirement for the intact 5'-regulatory domain in the viral replicative cycle. Interestingly, the addition of one extra nucleotide to the 3' end totally abolished the CAT activity indicating that an exact 3' terminus is critical in this process.

Phosphorylation of Sendai virus phosphoprotein by cellular protein kinase C zeta.

The phosphoproteins (P) of nonsegmented negative strand RNA viruses are viral RNA polymerase subunits involved in both transcription and replication during the virus life cycle. Phosphorylation of P proteins in several negative strand RNA viruses by specific cellular kinases was found to be required for P protein function. In the present study, using bacterially expressed unphosphorylated P protein of Sendai virus, a mouse parainfluenza virus, we have shown that the major cellular kinase that phosphorylates P protein in vitro is biochemically and immunologically indistinguishable from protein kinase C (PKC) zeta isoform. PKC zeta was packaged into the Sendai virion and remained associated with purified viral ribonucleoprotein, where it phosphorylated both the P and the nucleocapsid protein in vitro. When PKC zeta-specific inhibitory pseudosubstrate peptide was introduced into LLC-MK2 cells prior to Sendai virus infection, production of progeny virus was dramatically attenuated, and kinetic analysis revealed that primary transcription was repressed. These data indicate that phosphorylation of the Sendai virus P protein by PKC zeta plays a critical role in the virus life cycle.

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds.

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of Mr 32,000 each was discernible. Km values for NAD+ and myo-inositol 1-phosphate have been recorded as 2.8 X 10(-4) and 5.0 X 10(-4) M, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD+ are reduced.

Interaction of L and NS proteins of vesicular stomatitis virus with its template ribonucleoprotein during RNA synthesis in vitro.

Soluble transcriptase containing the L and the NS proteins was isolated from purified vesicular stomatitis virus and its binding with the template ribonucleoprotein containing the N protein-RNA complex was studied with respect to its ability to initiate and synthesize RNA in vitro. By using u.v.-irradiated template reconstituted with soluble transcriptase, it was shown that the synthesis of leader RNA and other small initiated mRNA sequences continued while full-length mRNA synthesis decreased by 90%. In the presence of ATP and CTP, the reconstituted complex synthesized polyphosphorylated oligonucleotides which include AC, AAC and AACA which represent 5'-terminal sequences transcribed from the leader template and genes coding for mRNAs. In the presence of arabinosyl ATP, an inhibitor of RNA synthesis in vitro, the synthesis of leader RNA was found to be inhibited considerably more than other small initiated mRNA sequences. Reconstitution of RNA synthesis with soluble transcriptase and template in the presence of viral matrix (M) protein at low ionic condition resulted in virtual cessation of leader RNA synthesis, although the synthesis of small initiated N mRNA, 11 to 14 bases, continued. These results suggest that transcriptase can bind at multiple sites on the genome template and initiate RNA chains.

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Role of cellular actin in human parainfluenza virus type 3 genome transcription.

The transcribing ribonucleoprotein (RNP) complex of human parainfluenza virus type 3 (HPIV-3) requires cellular actin for transcription of viral genome in vitro (De, B. P., Lesoon, A., and Banerjee, A. K. (1991) J. Virol. 65, 3268-3275). In this communication, we have studied the interactions between different molecular forms of actin and the RNP of HPIV-3 to understand the role of actin in mRNA synthesis. We demonstrate that both polymeric and monomeric forms of actin (obtained by DNase I treatment) bind strongly to the RNP at 100 mM KCl concentration (polymerizing buffer). The binding was virtually abolished at zero KCl concentration (depolymerizing buffer). Isolation of the RNP-actin complex and subsequent use in a transcription reaction showed that the bound actin alone was sufficient for mRNA synthesis in vitro. Interestingly, the DNase I-arrested monomeric form of actin failed to activate mRNA synthesis, indicating a requirement of polymerization of the bound actin during HPIV-3 transcription. Electron microscopic analyses revealed that a drastic structural modification of the RNP occurred because of the polymerization of actin from a loosely coiled and irregular structure to a condensed and flexible structure. Activation of transcription was observed also with poly-L-glutamic acid, a highly acidic polypeptide. However, unlike cellular actin, poly-L glutamic acid was able to activate only 10% of the input RNP. These results suggest that cellular actin activates HPIV-3 transcription by polymerizing specifically on the RNP complex. This event results in an alteration of the RNP structure that enhances its suitability for efficient transcription. The acidic domain of actin may play an important role in this process.

Human parainfluenza virus type 3 transcription in vitro: role of cellular actin in mRNA synthesis.

Purified ribonucleoprotein complexes of human parainfluenza virus type 3 (HPIV-3) virions required, in addition to the viral proteins, soluble cytoplasmic proteins from uninfected cells for the synthesis of mRNAs in vitro. In contrast to Sendai virus transcription, in vitro RNA synthesis from HPIV-3 ribonucleoprotein complexes was not stimulated significantly by purified tubulin. Moreover, cytoplasmic extract depleted of tubulin by immunoprecipitation stimulated HPIV-3 transcription effectively, suggesting involvement of a host protein(s) other than tubulin in the HPIV-3 transcription process. The transcription stimulatory factor was purified from uninfected cell extract by conventional chromatography and was found to contain a major 43-kDa polypeptide. In Western blot (immunoblot) analysis, this protein reacted with antiactin antibody, suggesting that the 43-kDa polypeptide is actin. This possibility was further supported by its polymerization activity and properties of binding to blue-Sepharose and heparin-Sepharose columns. Furthermore, when the cell extract was depleted of actin by immunoprecipitation by antiactin antibody, the stimulatory activity was abolished, indicating an involvement of actin in the stimulation of HPIV-3 transcription. After purification from RNAses, similar stimulatory activity associated with the 43-kDa protein was detected in other cell lines as well, including CV-1, HeLa, and BHK.

In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus.

The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [gamma-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[gamma-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by the L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.

Role of cellular kinases in the gene expression of nonsegmented negative strand RNA viruses.

Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs. The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor or transactivator of the polymerase. Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated. Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied. Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription. These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent.