Kinetic energy distributions of atomic ions from disintegration of argon containing nanoclusters in moderately intense nanosecond laser fields: Coulomb explosion or hydrodynamic expansion.

We report kinetic energies (KE) of multiply charged atomic ions (MCAI) from interactions of moderately intense nanosecond lasers at 532 nm with argon containing clusters, including neat and doped clusters with a trace amount of trichlorobenzene. We develop a mathematical method to retrieve speed and thereby kinetic energy information from analyzing the time-of-flight profiles of the MCAI. This method should be generally applicable in detections of energetic charged particles with high velocities, a realm where velocity map imaging is inadequate. From this analysis, we discover that the KE of MCAI from doped clusters demonstrates a quadratic dependence on the charge of the atomic ions, while for neat clusters, the dependence is cubic. This result confirms the nature of the cluster disintegration process to be dominated by Coulomb explosion. This result bears more similarity to reports from extreme vacuum ultraviolet (EUV) fields with similar intensities, than to reports from near infrared (NIR) intense laser fields. However, the charge state distribution from our experiment is the opposite: we observe more higher charge state ions than reported in EUV fields, and our charge state distribution is actually similar to those reported in NIR fields. We also report a significant effect of the external electric field on the charge state distribution of the atomic ions: the presence of an electric field can significantly increase the charge from the atomic ions, as shown by a three-fold reduction in the average kinetic energy per charge. Although molecular dynamics simulations have been implemented for experiments in the EUV and NIR, our results allude to the need of a concerted effort in this regime of moderately intense nanosecond laser fields. The significant decrease in charge state distribution and the significant increase in KE from doped clusters, compared with neat clusters, is a telltale sign that the true interaction time between the laser field and the cluster may be substantially shorter than the duration of the laser, a welcome relief for molecular dynamics simulations.

Neosuberitenone, a New Sesterterpenoid Carbon Skeleton; New Suberitenones; and Bioactivity against Respiratory Syncytial Virus, from the Antarctic Sponge Suberites sp.

Respiratory syncytial virus (RSV) is a highly contagious human pathogen that poses a significant threat to children under the age of two, and there is a current need for new small molecule treatments. The Antarctic sponge Suberites sp. is a known source of sesterterpenes, and following an NMR-guided fractionation procedure, it was found to produce several previously unreported metabolites. Neosuberitenone (1), with a new carbon scaffold herein termed the 'neosuberitane' backbone, six suberitenone derivatives (2-7), an ansellane-type terpenoid (8), and a highly degraded sesterterpene (9), as well as previously reported suberitenones A (10) and B (11), were characterized. The structures of all of the isolated metabolites including absolute configurations are proposed on the basis of NMR, HRESIMS, optical rotation, and XRD data. The biological activities of the metabolites were evaluated in a range of infectious disease assays. Suberitenones A, B, and F (3) were found to be active against RSV, though, along with other Suberites sp. metabolites, they were inactive in bacterial and fungal screens. None of the metabolites were cytotoxic for J774 macrophages or A549 adenocarcinoma cells. The selectivity of suberitenones A, B, and F for RSV among other infectious agents is noteworthy.

Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.

Human Respiratory Syncytial Virus NS2 Protein Induces Autophagy by Modulating Beclin1 Protein Stabilization and ISGylation.

Paramyxoviruses such as respiratory syncytial virus (RSV) are the leading cause of pneumonia in infants, the elderly, and immunocompromised individuals. Understanding host-virus interactions is essential for the development of effective interventions. RSV induces autophagy to modulate the immune response. The viral factors and mechanisms underlying RSV-induced autophagy are unknown. Here, we identify the RSV nonstructural protein NS2 as the virus component mediating RSV-induced autophagy. We show that NS2 interacts and stabilizes the proautophagy mediator Beclin1 by preventing its degradation by the proteasome. NS2 further impairs interferon-stimulated gene 15 (ISG15)-mediated Beclin1 ISGylation and generates a pool of "hypo-ISGylated" active Beclin1 to engage in functional autophagy. Studies with NS2-deficient RSV revealed that NS2 contributes to RSV-mediated autophagy during infection. The present study is the first report to show direct activation of autophagy by a paramyxovirus nonstructural protein. We also report a new viral mechanism for autophagy induction wherein the viral protein NS2 promotes hypo-ISGylation of Beclin1 to ensure availability of active Beclin1 to engage in the autophagy process. IMPORTANCE Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. Here, we identify the RSV nonstructural protein 2 (NS2) as the virus component involved in autophagy induction. NS2 promotes autophagy by interaction with and stabilization of the proautophagy mediator Beclin1 and by impairing its ISGylation to overcome autophagy inhibition. To the best of our knowledge, this is the first report of a viral protein regulating the autophagy pathway by modulating ISGylation of autophagy mediators. Our studies highlight a direct role of a paramyxovirus nonstructural protein in activating autophagy by interacting with the autophagy mediator Beclin1. NS2-mediated regulation of the autophagy and ISGylation processes is a novel function of viral nonstructural proteins to control the host response against RSV.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells.

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110-183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M's RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.

Use of Minigenome Systems to Study RSV Transcription.

Minigenome assays have been essential tools in the understanding of viral transcription and RNA replication for respiratory syncytial virus (RSV). Here, we describe the RSV minigenome assay for determining transcription by the viral polymerase in the absence of infection. We detail two different methods of detecting viral RNA synthesis: a firefly luciferase assay for rapid and sensitive measurement of RSV polymerase activity; and a real-time quantitative PCR method for determination of specific effects on the transcription of individual viral genes and the polar transcription gradient of RSV.

Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses.

UNLABELLED: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. IMPORTANCE: RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.

The interactome of the human respiratory syncytial virus NS1 protein highlights multiple effects on host cell biology.

Viral proteins can have multiple effects on host cell biology. Human respiratory syncytial virus (HRSV) nonstructural protein 1 (NS1) is a good example of this. During the virus life cycle, NS1 can act as an antagonist of host type I and III interferon production and signaling, inhibit apoptosis, suppress dendritic cell maturation, control protein stability, and regulate transcription of host cell mRNAs, among other functions. It is likely that NS1 performs these different roles through interactions with multiple host cell proteins. To investigate this and identify cellular proteins that could interact with NS1, we used quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis. This analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1. Specific immunoprecipitation using the GFP trap was used to confirm the ability of selected cellular proteins to interact individually with NS1. Infection of A549 cells with recombinant viruses deficient in the expression of NS1 and overexpression analysis both demonstrated that NS1 was necessary and sufficient for the enrichment of cells in the G(1) phase of the cell cycle.

The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism.

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin beta1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.

Human respiratory syncytial virus nonstructural protein NS2 antagonizes the activation of beta interferon transcription by interacting with RIG-I.

A wide variety of RNA viruses have been shown to produce proteins that inhibit interferon (IFN) production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to block IFN signaling by causing the proteasomal degradation of STAT2. In addition, recombinant RSVs lacking either NS1 or NS2 induce more IFN production than wild-type (wt) RSV in infected cells. However, the mechanisms by which the NS proteins perform this function are unknown. In this study, we focused on defining the mechanism by which NS2 inhibits the induction of IFN transcription. We find that NS2 is required for the early inhibition of IFN transcription since the infection of cells with NS2-deletion RSV resulted in a higher level of IRF3 activation at early time points postinfection compared with that of wt or NS1-deletion RSV infection. In addition, NS2 expression inhibits IFN transcription induced by both the RIG-I and TLR3 pathways. Furthermore, we show that NS2 inhibits RIG-I-mediated IFN promoter activation by binding to the N-terminal CARD of RIG-I and inhibiting its interaction with the downstream component MAVS (IPS-1, VISA, Cardif). Thus, the RSV NS2 protein is a multifunctional IFN antagonist that targets specific components of both the IFN induction and IFN signaling pathways.

Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1.

We report here the first biochemical and structural characterization of the respiratory syncytial virus (RSV) NS1 protein. We have used a pET-ubiquitin expression system to produce respiratory syncytial virus (RSV) NS1 protein in E. coli that contains a hexahistidine-tag on either the amino- or carboxyl-terminus (His(6)-NS1 and NS1-His(6), respectively). We have been able to isolate milligram quantities of highly purified His(6)-NS1 and NS1-His(6) by nickel affinity chromatography. Generation of recombinant RSV indicated that addition of the hexahistidine tag to the C-terminus of NS1 slightly decreased viral replication competence whereas addition of the tag to the N-terminus had no observable effect. Therefore, we performed a comprehensive biochemical and biophysical characterization on His(6)-NS1. His(6)-NS1 is monodisperse in solution as determined by dynamic light scattering analysis. Both gel filtration and analytical ultracentrifugation showed that His(6)-NS1 is predominantly a monomer. In agreement with theoretical predictions, circular dichroism spectroscopy showed that His(6)-NS1 contains 21% alpha-helices, 34% beta-sheets, and 45% undefined structure. Immunization with purified His(6)-NS1 generated an antiserum that specifically recognizes NS1 by immunoprecipitation from HEp-2 cells infected by RSV, indicating that His(6)-NS1 resembles native NS1. The availability of purified RSV NS1 will permit biochemical and structural investigations providing insight into the function of NS1 in viral replication and interferon antagonism.

Replacement of the respiratory syncytial virus nonstructural proteins NS1 and NS2 by the V protein of parainfluenza virus 5.

Paramyxoviruses have been shown to produce proteins that inhibit interferon production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to have interferon antagonist activity through an unknown mechanism. To understand further the functions of NS1 and NS2, we generated recombinant RSV in which both NS1 and NS2 were replaced by the PIV5 V protein, which has well-characterized IFN antagonist activities (DeltaNS1/2-V). Expression of V was able to partially inhibit IFN responses in DeltaNS1/2-V-infected cells. In addition, the replication kinetics of DeltaNS1/2-V were intermediate between DeltaNS1/2 and wild-type (rA2) in A549 cells. However, expression of V did not affect the ability of DeltaNS1/2-V to activate IRF3 nuclear translocation and IFNbeta transcription. These data indicate that V was able to replace some of the IFN inhibitory functions of the RSV NS1 and NS2 proteins, but also that NS1 and NS2 have functions in viral replication beyond IFN antagonism.

Function of the respiratory syncytial virus small hydrophobic protein.

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, encodes a small hydrophobic (SH) protein of unknown function. Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, also encodes an SH protein, which inhibits tumor necrosis factor alpha (TNF-alpha) signaling. In this study, recombinant PIV5 viruses without their own SH but containing RSV SH (from RSV strain A2 or B1) in its place (PIV5DeltaSH-RSV SH) and RSV lacking its own SH (RSVDeltaSH) were generated and analyzed. The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-alpha signaling.

Deletion of M2 gene open reading frames 1 and 2 of human metapneumovirus: effects on RNA synthesis, attenuation, and immunogenicity.

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [rdeltaM2-1, rdeltaM2-2, and rdeltaM2(1+2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The rdeltaM2-2 mutants exhibited a two- to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with rdeltaM2-1 or rdeltaM2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with rdeltaM2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV rdeltaM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.

Effects of nonstructural proteins NS1 and NS2 of human respiratory syncytial virus on interferon regulatory factor 3, NF-kappaB, and proinflammatory cytokines.

Human respiratory syncytial virus (HRSV) is the leading cause of serious pediatric acute respiratory tract infections, and a better understanding is needed of the host response to HRSV and its attenuated vaccine derivatives. It has been shown previously that HRSV nonstructural proteins 1 and 2 (NS1 and NS2) inhibit the induction of alpha/beta interferon (IFN-alpha/beta) in A549 cells and human macrophages. Two principal transcription factors for the early IFN-beta and -alpha1 response are interferon regulatory factor 3 (IRF-3) and nuclear factor kappaB (NF-kappaB). At early times postinfection, wild-type HRSV and the NS1/NS2 deletion mutants were very similar in the ability to activate IRF-3. However, once NS1 and NS2 were expressed significantly, they acted cooperatively to suppress activation and nuclear translocation of IRF-3. Since these viruses differed greatly in the induction of IFN-alpha/beta, NF-kappaB activation was evaluated in Vero cells, which lack the structural genes for IFN-alpha/beta and would preclude confounding effects of IFN-alpha/beta. This showed that deletion of the NS2 gene sharply reduced the ability of HRSV to induce activation of NF-kappaB. Since recombinant HRSVs from which the NS1 or NS2 genes have been deleted are being developed as vaccine candidates, we investigated whether the changes in activation of host transcription factors and increased IFN-alpha/beta production had an effect on the epithelial production of proinflammatory factors. Viruses lacking NS1 and/or NS2 stimulated modestly lower production of RANTES (Regulated on Activation Normal T-cell Expressed and Secreted), interleukin 8, and tumor necrosis factor alpha compared to wild-type recombinant RSV, supporting their use as attenuated vaccine candidates.

Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate.

Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The DeltaSH, DeltaG, and DeltaSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the DeltaG and DeltaSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the DeltaG and DeltaSH/G viruses represent promising vaccine candidates. In contrast, DeltaSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected].

Wild-type human respiratory syncytial virus (HRSV) is a poor inducer of alpha/beta interferons (IFN-alpha/beta). However, recombinant HRSV lacking the NS1 and NS2 genes (Delta NS1/2) induced high levels of IFN-alpha and -beta in human pulmonary epithelial cells (A549) as well as in macrophages derived from primary human peripheral blood monocytes. Results with NS1 and NS2 single- and double-gene-deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role. The relative contributions of the individual NS proteins were the converse of that recently described for bovine RSV (J. F. Valarcher, J. Furze, S. Wyld, R. Cook, K. K. Conzelmann, and G. Taylor, J. Virol. 77:8426-8439, 2003). This pattern of inhibition by HRSV NS1 and NS2 also extended to the newly described antiviral cytokines IFN-lambda 1, -2 and -3.

Effects of altering the transcription termination signals of respiratory syncytial virus on viral gene expression and growth in vitro and in vivo.

Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.