The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production.

UNLABELLED: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE: We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.

The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.

Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees.

Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2DeltaNS1 and rA2DeltaM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2DeltaNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2DeltaNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2DeltaM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2DeltaNS1 and rA2DeltaM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2DeltaNS1 and rA2DeltaM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the cpts248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2DeltaNS1 or rA2DeltaM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.

NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs.

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.

Rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics.

RSV is a major cause of pediatric respiratory tract disease worldwide, but a vaccine is not yet available. It is now possible to prepare live infectious RSV completely from cDNA. This provides a method for introducing defined mutations into infectious virus, making possible the rational design of a live-attenuated vaccine virus for intranasal administration. This is particularly important for RSV, for which achieving the appropriate balance between attenuation and immunogenicity by conventional methods has proven elusive. We took advantage of the existence of a panel of biologically derived vaccine candidate viruses that were incompletely attenuated but well characterized biologically. The mutations in these viruses were identified by sequence analysis and characterized by insertion into recombinant virus, thereby providing a menu of known attenuating mutations. These included a series of amino acid point mutations, mostly in the L polymerase, and a nucleotide substitution in a transcription gene-start signal, a cis-acting RNA element. The second source of mutations was from experimental mutational analysis of recombinant virus and involves deletion of the NS1, NS2, or SH gene. We have reconstructed a previously tested, biologically derived attenuated virus, cpts248/404, in recombinant form and are now proceeding to introduce additional mutations from the menu to achieve stepwise increases in attenuation. The ability to modify the attenuation phenotype incrementally in a directed manner should result in an appropriate vaccine virus.

Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees.

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.

Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein.

The second gene in the 3'-to-5' gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (DeltaNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and DeltaNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the DeltaNS2 virus. Synthesis of readthrough mRNAs was affected only for the DeltaNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant DeltaNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the DeltaNS2 RSV is a candidate for vaccine development.

Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles.

We developed a system to identify the viral proteins required for the packaging and passage of human respiratory syncytial virus (RSV) by reconstructing these events with cDNA-encoded components. Plasmids encoding individual RSV proteins, each under the control of a T7 promoter, were cotransfected in various combinations together with a plasmid containing a minigenome into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Supernatants from these cells were passaged onto fresh cells which were then superinfected with RSV. Functional reconstitution of RSV-specific packaging and passage was detected by expression of the reporter gene carried on the minigenome. As expected, the four nucleocapsid proteins N, P, L, and M2-1 failed to direct packaging and passage of the minigenome. Passage was achieved by further addition of plasmids expressing three membrane-associated proteins, M, G, and F; inclusion of the fourth envelope- associated protein, SH, did not alter passage efficiency. Passage was reduced 10- to 20-fold by omission of G and was abrogated by omission of either M or F. Coexpression of the nonstructural NS1 or NS2 protein had little effect on packaging and passage except through indirect effects on RNA synthesis in the initial transfection. The M2-1 transcription elongation factor was not required for the generation of passage-competent particles. However, addition of increasing quantities of M2-1 to the transfection mediated a dose-dependent inhibition of passage which was alleviated by coexpression of the putative negative regulatory factor M2-2. Omission of the L plasmid reduced passage 10- to 20-fold, most likely due to reduced availability of encapsidated minigenomes for packaging. However, the residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles. Omission of N or P from the transfection abrogated passage. Thus, the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G.

A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome.

Persistent infection of C3H/St mice with certain strains of lymphocytic choriomeningitis virus (LCMV) causes a growth hormone (GH) deficiency syndrome (GHDS) manifested as growth retardation and hypoglycemia. Infected mice show high levels of viral replication in the GH-producing cells in the anterior pituitary leading to decreased synthesis of GH mRNA and protein despite the absence of detectable virus-induced cell structural damage. Virus clones isolated from the GHDS-negative LCMV WE strain can cause the disease, while others cannot. The genetic basis of this phenotypic difference is a nucleotide substitution resulting in a single amino acid difference in the viral glycoprotein. Reassortant studies indicate that the single amino acid substitution (Ser-153 to Phe) is sufficient to allow infection of the GH-producing cells and cause GHDS. These results show that a single change in the genome can affect viral pathogenicity by altering the tropism of the virus.

Variants able to cause growth hormone deficiency syndrome are present within the disease-nil WE strain of lymphocytic choriomeningitis virus.

Persistent infection of C3H/St mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong leads to disordered growth and hypoglycemia. Both host and viral determinants contribute to this growth hormone (GH) deficiency syndrome (GHDS). Development of the GHDS correlates with the virus's ability to replicate in the GH-producing cells and cause reduced levels of GH synthesis. LCMV strain WE infects few GH-producing cells and does not cause GHDS in C3H/St mice. We show here that clonal variants isolated from the GHDS-nil WE population are able to replicate at high levels in GH-producing cells and cause GHDS in C3H/St mice. These variants are stably maintained, but phenotypically silent, within the GHDS-nil WE population.

Suppression of lymphocytic choriomeningitis virus--induced growth hormone deficiency syndrome by disease-negative virus variants.

Populations of RNA viruses consist of heterogeneous mixtures of related genomes (quasispecies). Isolation of variants present at low levels within a population can result in clonal virus populations which display markedly different phenotypes upon infection of the host. The mechanisms by which these variants are maintained within the original quasispecies are not understood. Certain strains of lymphocytic choriomeningitis virus (LCMV) cause a growth hormone deficiency syndrome (GHDS) when inoculated into newborn C3H/St mice while others do not. We have previously described the isolation of virus clones from the GHDS-negative WE strain of LCMV which differ in their ability to cause GHDS. To investigate how disease-positive clones can remain hidden within a disease-negative parental population, we examined whether infection with mixtures of the GHDS-negative (WE c54) and GHDS-positive (WE c2.5) clones could cause GHDS. Neonatal C3H/ST mice infected with 100:1 or 10:1 ratios of WE c54 to WE c2.5 did not develop the syndrome, while animals infected with 1:1 or lower ratios did. Development of GHDS correlated with the extent to which the GH-producing cells of the anterior pituitary were infected. These data indicate that a large excess of disease-negative clones can restrict the replication of disease-positive clones in GH-producing cells, thus preventing the onset of GHDS. In addition, our results indicate that a threshold for phenotypic dominance exists. Interestingly, WE c54 did not entirely outcompete WE c2.5 in mice infected with the 100:1 ratio, suggesting a mechanism whereby pathogenic viruses can be maintained within a nonpathogenic viral population.

Experimental reproduction of severe hypoglycemia and spiking mortality syndrome using field-derived and embryo-passaged preparations.

The clinical signs, enteritis, weight depression, and hypoglycemia of spiking mortality syndrome were experimentally reproduced in broiler breeders and broiler chicks. Inocula included 1) virus-like particles from intestines of chicks with spiking mortality syndrome that had been banded in a discontinuous Renograffin gradient, 2) homogenized darkling beetles collected from litter of farms where spiking mortality syndrome had occurred repeatedly, and 3) homogenized embryos which had been inoculated with the Renograffin-banded material. Arkansas variant infectious bronchitis virus and arenavirus-like particles were identified in the inocula. Serology on samples from surviving chicks suggested the presence of an avian encephalomyelitis virus in one of the inocula. One-day-old (n = 172) and 2.5-day-old (n = 30) chicks were inoculated orally, and some were also injected intraperitoneally or subcutaneously, with 0.5 ml of the inocula. Twelve to fourteen days postinoculation, chicks were fasted for 4-6 hours, then briefly stressed with a cool water spray. Within 1.5 hours, inoculated chicks began dying with severe hypoglycemia and clinical signs of spiking mortality syndrome. Body weights were significantly depressed. Uninoculated controls (n = 130) from the same hatches, also fasted and stressed, were unaffected clinically and were not hypoglycemic. One group (n = 52) of inoculated chicks exposed to a controlled lighting program was unaffected clinically, had significantly higher mean plasma glucose levels, and had significantly less body weight depression than chicks exposed to continuous lighting. We concluded that exposure to controlled amounts of light/darkness can ameliorate much of the hypoglycemia, mortality, and runting-stunting associated with spiking mortality syndrome of chickens. The significance of the viruses and virus-like particles detected in the inocula is currently under investigation.

Prevention of runting and cachexia by a chimeric TNF receptor-Fc protein.

Tumor necrosis factor alpha (TNF) is an important mediator of septic shock and cachexia. A soluble form of the human type 2 TNF receptor, constructed by joining the Fc region of human IgG1 to the TNF receptor, prevents weight loss in nude mice bearing a TNF-secreting tumor. This soluble receptor was also used to treat TNF transgenic mice which were runting and died before reaching reproductive age. After continuous treatment with soluble TNF receptor, the TNF transgenic mice grew to normal size and reproduced. Thus, soluble TNF may be useful in counteracting the detrimental systemic effects of TNF in a clinical setting.

Alternatively spliced ltk mRNA in neurons predicts a receptor with a larger putative extracellular domain.

Ltk is a new member of the ros/insulin receptor family of tyrosine kinases that is expressed in murine B-lymphocyte precursors and forebrain neurons. We previously reported that lymphoid ltk cDNAs predict a 69 kDa transmembrane glycoprotein, which uses a CUG translational start codon and has a 110 amino acid putative extracellular domain. We now show that the predominant ltk mRNA in brain is alternatively spliced and predicts a protein with a substantially larger extracellular part. The human ltk gene maps to chromosome 15, bands q13-21, a region containing the breakpoint of a recurring chromosomal abnormality in B-cell non-Hodgkin lymphomas.

Long-term inhibition of tumor growth by tumor necrosis factor in the absence of cachexia or T-cell immunity.

The relationship between detrimental (cachectic) and beneficial (antitumor) effects of tumor necrosis factor (TNF) was studied in mice bearing murine tumors transfected to secrete human TNF. In vitro, the TNF-producing transfectants were resistant to the secreted TNF and grew at rates similar to those of untransfected cells or transfected cells that did not secrete TNF. However, tumors formed by the TNF-secreting cells in vivo remained much smaller than the nonsecreting (transfected and untransfected) tumors. This inhibition of tumor growth required only relatively low serum levels of TNF, persisted for many weeks, and was independent of T cells since it occurred in nude mice. Growth of the TNF-secreting tumors increased dramatically after treatment with anti-human TNF antibody, indicating that extracellular TNF secreted by the tumor cells was necessary for the tumor inhibition. Severe weight loss characteristic of cachexia only occurred in animals with very high serum TNF levels (250 pg/ml) and could be prevented or reversed by anti-TNF antibody treatment. These data are consistent with the existence of a therapeutic window in which persistent exposure to human TNF can lead to prolonged inhibition of tumor growth in the absence of T-cell immunity or severe weight loss and without development of resistant tumor variants.