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.

Interferon action against human parainfluenza virus type 3: involvement of a novel antiviral pathway in the inhibition of transcription.

Interferon (IFN)-induced 2'-5' oligoadenylate synthetase (2-5A synthetase)/RNase L, PKR, and Mx proteins are considered to be the principal antiviral protein pathways through which IFN induces an antiviral state. It was previously reported that human parainfluenza virus type 3 (HPIV3) multiplication was inhibited by IFN-alpha in human lung epithelial cells A549 and that MxA was found to contribute to the inhibition process (Zhao et al., Virology 220:330-338, 1996). Viral primary transcription was dramatically inhibited in A549 cells after IFN-alpha treatment, but a step following primary transcription was inhibited in U87-MxA cells constitutively expressing MxA. Here we have investigated the role of MxA, believed to be cell type specific, and other antiviral pathways in the inhibition of viral primary transcription. Our data indicate that a novel IFN-induced pathway(s) is involved in the inhibition of primary transcription. This is based on the following findings: (i) IFN-alpha inhibited viral primary transcription in U87-MxA and other cell types including cells lacking MxA; (ii) cells constitutively expressing 2-5A synthetase had no antiviral effect against HPIV3; and (iii) primary transcription occurred in the absence of protein synthesis, a step of PKR target. The novel antiviral pathway(s) was induced by both IFN-alpha and IFN-gamma to establish an effective antiviral state against HPIV3. By using IFN-alpha-signaling mutant cells, we found that IFN-gamma could elicit antiviral effect against HPIV3 without cross talk with the IFN-alpha-signaling pathway. These data provide the first evidence that a novel antiviral pathway(s) contributes to the antiviral action of IFN against a nonsegmented negative-strand RNA virus by targeting the primary transcription.

Human parainfluenza virus type 3 inhibits gamma interferon-induced major histocompatibility complex class II expression directly and by inducing alpha/beta interferon.

Human parainfluenza virus type 3 (HPIV3) is one of the major causes of bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity involving major histocompatibility complex (MHC) class I and class II molecules plays an important role in controlling virus infection. Several viruses have been shown to down-regulate gamma interferon (IFN-gamma)-mediated MHC class II expression. In this communication, we show that HPIV3 strongly inhibits the IFN-gamma-induced MHC class II expression in HT1080 human fibrosarcoma cells. The culture supernatant of HPIV3-infected cells also inhibited IFN-gamma-induced MHC class II expression, a phenomenon that was found to be due, in large part, to alpha/beta interferon (IFN-alpha/beta). Expression of MHC class I and intercellular adhesion molecule 1 occurred efficiently in cells simultaneously infected with HPIV3 and treated with IFN-gamma, indicating that the inhibitory effect of HPIV3 was specific to MHC class II. STAT1 activation was not affected by HPIV3 at early postinfection times but was partially inhibited at later times. These data suggested that the potent inhibition of MHC class II expression was, in major part, due to a defect downstream of STAT1 activation in the IFN-gamma-induced MHC class II expression pathway. Class II transactivator (CIITA) is the unique mediator of IFN-gamma-induced transcription from the MHC class II promoter. By RNase protection analysis, CIITA expression was found to be strongly inhibited in HPIV3-infected cells. The culture supernatant containing IFN-alpha/beta, on the other hand, inhibited MHC class II expression without affecting STAT1 and CIITA expression. These data indicate that HPIV3 inhibits IFN-gamma-induced MHC class II expression primarily by the viral gene products targeting CIITA and additionally by inducing IFN-alpha/beta to target one or more steps further downstream.

Role of NH(2)- and COOH-terminal domains of the P protein of human parainfluenza virus type 3 in transcription and replication.

The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.

Specific phosphorylated forms of glyceraldehyde 3-phosphate dehydrogenase associate with human parainfluenza virus type 3 and inhibit viral transcription in vitro.

We previously reported specific interaction of cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the key glycolytic enzyme, and La protein, the RNA polymerase III transcription factor, with the cis-acting RNAs of human parainfluenza virus type 3 (HPIV3) and packaging of these proteins within purified virions (B. P. De, S. Gupta, H. Zhao, J. Z. Drazba, and A. K. Banerjee, J. Biol. Chem. 271:24728-24735, 1996). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [(32)P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3' genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viral cis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression.

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.

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 up-regulates major histocompatibility complex class I and II expression on respiratory epithelial cells: involvement of a STAT1- and CIITA-independent pathway.

Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infection-related damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility complex (MHC) class I and II molecules on human lung epithelial (A549) and epithelium-like (HT1080) cells following HPIV3 infection. MHC class I was induced by HPIV3 in these cells at levels similar to those observed with natural inducers such as beta and gamma interferon (IFN-beta and -gamma). MHC class II was also efficiently induced by HPIV3 in these cells. UV-irradiated culture supernatants from infected cells were able to induce MHC class I but not MHC class II, suggesting involvement of released factors for the induction of MHC class I. Quantitation of IFN types I and II in the culture supernatant showed the presence of IFN-beta as the major cytokine, while IFN-gamma was undetectable. Anti-IFN-beta, however, blocked the HPIV3-mediated induction of MHC class I only partially, indicating that viral antigens, besides IFN-beta, are directly involved in the induction process. The induction of MHC class I and class II directed by the viral antigens was confirmed by using cells lacking STAT1, an essential intermediate of the IFN signaling pathways. HPIV3 induced both MHC class I and class II molecules in STAT1-null cells. Furthermore, MHC class II was also induced by HPIV3 in cells defective in class II transactivator, an important intermediate of the IFN-gamma-mediated MHC class II induction pathway. Together, these data indicate that the HPIV3 gene product(s) is directly involved in the induction of MHC class I and II molecules. The induction of MHC class I and II expression by HPIV3 suggests that it plays a role in the infection-related immunity and pathogenesis.

Involvement of actin microfilaments in the replication of human parainfluenza virus type 3.

Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.

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.

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.

Phosphorylation of canine distemper virus P protein by protein kinase C-zeta and casein kinase II.

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-zeta) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-zeta has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-zeta has only minor activity. Selective inhibition of PKC-zeta activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-zeta in the virus replication cycle. The broad tissue expression of PKC-zeta parallels the pantropic nature of CDV infections, suggesting that PKC-zeta activity is a determinant of cellular permissiveness to CDV replication.

Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cis-acting RNAs of human parainfluenza virus type 3.

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.

Inhibition of human parainfluenza virus-3 replication by interferon and human MxA.

We have investigated the IFN-mediated inhibition of human parainfluenza virus-3 (HPIV-3) replication in cultured human A549 cells. IFN-alpha inhibited the virus yield significantly with concomitant reduction of viral RNA accumulation by more than 90%. Further studies indicated that the inhibitory action of IFN was at the level of primary transcription of HPIV3 replication. Since the IFN-inducible protein, MxA, has been shown to inhibit virus replication in several RNA viruses, we examined the role of MxA in HPIV-3 replication using a stably transfected human glioblastoma cell line expressing MxA. In these cells HPIV-3 replication was decreased by more than 100-fold depending on the virus dosage used with concomitant inhibition of viral RNA synthesis by about 80%. However, the viral primary transcription was not affected in this MxA-producing cell line. In contrast, in the parental cell line IFN-mediated inhibition occurred at the primary transcription step of HPIV-3 replication. These data suggest that in addition to MxA, other IFN-inducible proteins are involved in the anti-HPIV-3 effect of IFN in both the cell lines used.

Human parainfluenza virus type 3 phosphoprotein: identification of serine 333 as the major site for PKC zeta phosphorylation.

The human parainfluenza virus type 3 P protein is an RNA polymerase subunit involved in both transcription and replication during the life cycle of the virus. Our laboratory has recently shown that the P protein is phosphorylated both in vitro and in vivo by the cellular protein kinase C (PKC) isoform zeta and that this phosphorylation is essential for viral replication. To identify the site(s) of phosphorylation, we have used CNBr cleavage, phosphoamino acid analysis, and two-dimensional tryptic peptide mapping of the in vitro and in vivo phosphorylated P protein. We demonstrate that when bacterially expressed unphosphorylated P is labeled in vitro with either commercial PKC or purified recombinant PKC zeta P protein has one major phosphorylation site. By site-directed mutagenesis of PKC consensus sites in the P protein, the primary phosphorylation site is found to be Ser 333. The same site appeared to be modified when viral P protein was phosphorylated in vitro by the PKC packaged within the virion and in the P protein of progeny virion labeled in vivo.

Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication.

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.

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.