Cindy Luongo

Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse

Mutations in the human APC gene caused various familial colon cancer syndromes. The Multiple intestinal neoplasia (Min) mouse provides an excellent model for familial colon cancer: it carries a mutant mouse Apc gene and develops many intestinal adenomas. Here, we analyze how this tumor phenotype is dramatically modified by genetic background. We report the genetic mapping of a locus that strongly modifies tumor number in Min/+ animals. This gene, Mom-1 (Modifier of Min-1), maps to distal chromosome 4 and controls about 50% of genetic variation in tumor number in two intraspecific backcrosses. The mapping is supported by a LOD score exceeding 14. Interestingly, Mom-1 lies in a region of synteny conservation with human chromosome 1p35-36, a region of frequent somatic loss of heterozygosity in a variety of human tumors, including colon tumors. These results provide evidence of a major modifier affecting expression of an inherited cancer syndrome.

ApcMin: A mouse model for intestinal and mammary tumorigenesis

Min (multiple intestinal neoplasia) is a mutant allele of the murine Apc (adenomatous polyposis coli) locus, encoding a nonsense mutation at codon 850. Like humans with germline mutations in APC, Min/+ mice are predisposed to intestinal adenoma formation. The number of adenomas is influenced by modifier loci carried by different inbred strains. One modifier locus, Mom-1 (modifier of Min-1), maps to distal chromosome 4. Intestinal tumours from both B6 (C57BL/6J) and hybrid Min/+ mice show extensive loss of the wild-type allele at Apc. B6 Min/+ female mice are predisposed to spontaneous mammary tumours. The incidence of both intestinal and mammary tumours can be increased in an age-specific manner by treatment with ethylnitrosourea (ENU). Min mice provide a good animal model for studying the role of Apc and interacting genes in the initiation and progression of intestinal and mammary tumorigenesis.

A Chimeric A2 Strain of Respiratory Syncytial Virus (RSV) with the Fusion Protein of RSV Strain Line 19 Exhibits Enhanced Viral Load, Mucus, and Airway Dysfunction

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of respiratory failure and viral death in infants. Abundant airway mucus contributes to airway obstruction in RSV disease. Interleukin-13 (IL-13) is a mediator of pulmonary mucus secretion. It has been shown that infection of BALB/c mice with the RSV line 19 strain but not with the RSV A2 laboratory strain results in lung IL-13 and mucus expression. Here, we sequenced the RSV line 19 genome and compared it to the commonly used A2 and Long strains. There were six amino acid differences between the line 19 strain and both the A2 and Long RSV strains, five of which are in the fusion (F) protein. The Long strain, like the A2 strain, did not induce lung IL-13 and mucus expression in BALB/c mice. We hypothesized that the F protein of RSV line 19 is more mucogenic than the F proteins of A2 and Long. We generated recombinant, F-chimeric RSVs by replacing the F gene of A2 with the F gene of either line 19 or Long. Infection of BALB/c mice with RSV rA2 line 19F resulted in lower alpha interferon lung levels 24 h postinfection, higher lung viral load, higher lung IL-13 levels, greater airway mucin expression levels, and greater airway hyperresponsiveness than infection with rA2-A2F or rA2-LongF. We identified the F protein of RSV line 19 as a factor that plays a role in pulmonary mucin expression in the setting of RSV infection.

Alpha and Lambda Interferon Together Mediate Suppression of CD4 T Cells Induced by Respiratory Syncytial Virus

ABSTRACT The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [3H]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained alpha interferon (IFN-α; median, 43 pg/ml) and IFN-λ (approximately 1 to 20 ng/ml). Neutralization of IFN-α with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-λ with MAb against either of its receptor chains, IFN-λR1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of interferons.

RSV604, a Novel Inhibitor of Respiratory Syncytial Virus Replication

ABSTRACT Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.

Nonstructural Proteins 1 and 2 of Respiratory Syncytial Virus Suppress Maturation of Human Dendritic Cells

ABSTRACT Human respiratory syncytial virus (RSV) is the most important agent of serious pediatric respiratory tract disease worldwide. One of the main characteristics of RSV is that it readily reinfects and causes disease throughout life without the need for significant antigenic change. The virus encodes nonstructural protein 1 (NS1) and NS2, which are known to suppress type I interferon (IFN) production and signaling. In the present study, we monitored the maturation of human monocyte-derived myeloid dendritic cells (DC) following inoculation with recombinant RSVs bearing deletions of the NS1 and/or NS2 proteins and expressing enhanced green fluorescent protein. Deletion of the NS1 protein resulted in increased expression of cell surface markers of DC maturation and an increase in the expression of multiple cytokines and chemokines. This effect was enhanced somewhat by further deletion of the NS2 protein, although deletion of NS2 alone did not have a significant effect. The upregulation was largely inhibited by pretreatment with a blocking antibody against the type I IFN receptor, suggesting that suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins. Therefore, this study identified another effect of the NS1 and NS2 proteins. The observed suppression of DC maturation may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection.

Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity

ABSTRACT 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 [rΔM2-1, rΔM2-2, and rΔM2(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 rΔM2-2 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 rΔM2-1 or rΔM2(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 rΔM2-2, 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 rΔM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.

A gene deletion that up-regulates viral gene expression yields an attenuated RSV vaccine with improved antibody responses in children

A live attenuated vaccine candidate for RSV may decrease viral replication while enhancing immunogenicity in children. Outflanking RSV Respiratory syncytial virus (RSV) infection may lead to severe respiratory illness in young children. Researchers are working to develop a live attenuated vaccine, which would mimic the natural course of infection; however, inhibiting viral replication also limits the immune response. Now, Karron et al. report that a version of RSV lacking the M2-2 protein can induce immunity despite decreased vaccine virus shedding in young children. The lack of M2-2 resulted in decreased viral RNA replication needed for virus production while allowing gene transcription and antigen synthesis required for stimulating the immune response. Children who received the vaccine produced anti-RSV antibodies without medically attended illness in the subsequent RSV season, suggesting that this approach may provide protective immunity to RSV. Respiratory syncytial virus (RSV) is the leading viral cause of severe pediatric respiratory illness, and a safe and effective vaccine for use in infancy and early childhood is needed. We previously showed that deletion of the coding sequence for the viral M2-2 protein (ΔM2-2) down-regulated viral RNA replication and up-regulated gene transcription and antigen synthesis, raising the possibility of development of an attenuated vaccine with enhanced immunogenicity. RSV MEDI ΔM2-2 was therefore evaluated as a live intranasal vaccine in adults, RSV-seropositive children, and RSV-seronegative children. When results in RSV-seronegative children were compared to those achieved with the previous leading live attenuated RSV candidate vaccine, vaccine virus shedding was significantly more restricted, yet the postvaccination RSV-neutralizing serum antibody achieved [geometric mean titer (GMT) = 1:97] was significantly greater. Surveillance during the subsequent RSV season showed that several seronegative RSV MEDI ΔM2-2 recipients had substantial antibody rises without reported illness, suggesting that the vaccine was protective yet primed for anamnestic responses to RSV. Rational design appears to have yielded a candidate RSV vaccine that is intrinsically superior at eliciting protective antibody in RSV-naïve children and highlights an approach for the development of live attenuated RSV vaccines.

Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization

Significance Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded to increase the usage of underrepresented codon pairs, leaving amino acid coding unchanged. CPD viruses were temperature-sensitive and grew less efficiently in vitro than wild-type RSV. In addition, the CPD viruses exhibited a range of restriction in mice and African green monkeys that compared favorably with existing attenuated strains presently in clinical studies. This study produced examples of a new type of vaccine candidate for RSV and showed that CPD of a nonsegmented negative-strand RNA virus can rapidly generate vaccine candidates with a range of attenuation. Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.

Binding Site for S-Adenosyl-l-methionine in a Central Region of Mammalian Reovirus λ2 Protein EVIDENCE FOR ACTIVITIES IN mRNA CAP METHYLATION

One or more proteins in mammalian reovirus core particles mediate two RNA methylation activities, (guanosine-7-N)-methyltransferase and (guanosine-2′-O)-methyltransferase, that contribute to forming the 5′ cap 1 structure on viral mRNA. We used UV irradiation to identify core proteins that bindS-adenosyl-l-methionine (SAM), the methyl-group donor for both methyltransferases. A [methyl-3H]SAM-binding site was observed among the reovirus λ proteins; was shown to be specific by competition with low levels of S-adenosyl-l-homocysteine, the product of methyl-group transfer from SAM; and was subsequently localized to protein λ2. λ2 mediates the guanylyltransferase reaction in cap formation and was previously proposed to mediate one or both methylation reactions as well. SAM binding was demonstrated for both λ2 in cores and λ2 expressed in insect cells from a recombinant baculovirus. Using three different methods to cleave λ2, a binding site for SAM was tentatively localized to a central region of λ2, between residues 792 and 1100, which includes a smaller region with sequence similarity to the SAM-binding pocket of other methyltransferases. Alanine substitutions at positions 827 and 829 within this predicted binding region greatly reduced the capacity of baculovirus-expressed λ2 protein to undergo UV cross-linking to SAM but had no effects on either the guanylyltransferase activity of this protein or its conformation as judged by partial proteolysis, suggesting that one or both of these residues is essential for SAM binding. Based on these findings, we propose that the two methyltransferase activities involved in mRNA capping by reovirus cores utilize a single SAM-binding pocket within a central region of λ2.