Identification of a novel virulence factor in recombinant pneumonia virus of mice

Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (Delta G) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the Delta G mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.     

Mechanical ventilation enhances lung inflammation and caspase activity in a model of mouse pneumovirus infection

Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1alpha (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection.     

Degrons at the C terminus of the pathogenic but not the nonpathogenic hantavirus G1 tail direct proteasomal degradation

Pathogenic hantaviruses cause two human diseases: hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). The hantavirus G1 protein contains a long, 142-amino-acid cytoplasmic tail, which in NY-1 virus (NY-1V) is ubiquitinated and proteasomally degraded (E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow, J. Virol. 77: 10760-10768, 2003). Here we report that the G1 cytoplasmic tails of pathogenic Andes (HPS) and Hantaan (HFRS) viruses are also degraded by the proteasome and that, in contrast, the G1 tail of nonpathogenic Prospect Hill virus (PHV) is stable and not proteasomally degraded. We determined that the signals which direct NY-1V G1 tail degradation are present in a hydrophobic region within the C-terminal 30 residues of the protein. In contrast to that of PHV, the NY-1V hydrophobic domain directs the proteasomal degradation of green fluorescent protein and constitutes an autonomous degradation signal, or "degron," within the NY-1V G1 tail. Replacing 4 noncontiguous residues of the NY-1V G1 tail with residues present in the stable PHV G1 tail resulted in a NY-1V G1 tail that was not degraded by the proteasome. In contrast, changing a different but overlapping set of 4 PHV residues to corresponding NY-1V residues directed proteasomal degradation of the PHV G1 tail. The G1 tails of pathogenic, but not nonpathogenic, hantaviruses contain intervening hydrophilic residues within the C-terminal hydrophobic domain, and amino acid substitutions that alter the stability or degradation of NY-1V or PHV G1 tails result from removing or adding intervening hydrophilic residues. Our results identify residues that selectively direct the proteasomal degradation of pathogenic hantavirus G1 tails. Although a role for the proteasomal degradation of the G1 tail in HPS or HFRS is unclear, these findings link G1 tail degradation to viral pathogenesis and suggest that degrons within hantavirus G1 tails are potential virulence determinants.     

Pulmonary eosinophilia and production of MIP-1alpha are prominent responses to infection with pneumonia virus of mice

Human eosinophils secrete two distinct ribonucleases that have antiviral activity against pathogens of the family Paramyxoviridae. To examine the role of eosinophils and their ribonucleases in host defense against paramyxovirus pathogens in vivo, we have developed a mouse model involving a viral pathogen that naturally targets a rodent host. In this work we describe infection of Balb/c mice with pneumonia virus of mice (PVM, strain J3666), a paramyxovirus pathogen found frequently among rodent populations. We show here that pulmonary eosinophilia is an immediate response to infection with PVM, with bronchoalveolar lavage fluid containing 12-14% eosinophils obtained as early as day 3 postinoculation. Infection is accompanied by the production of macrophage inflammatory protein-1-alpha (MIP-1alpha), a chemokine that has been associated with the pulmonary eosinophilia observed in response to respiratory syncytial virus infection in humans and with enhanced clearance of influenza virus in mice. Interestingly, we observed no changes in expression of the chemoattractants eotaxin and RANTES in response to PVM infection, and interleukin-5 remained undetectable throughout. These responses-clinical pathology, viral recovery, pulmonary eosinophilia, and production of MIP-1alpha-will provide a means for exploring the role of eosinophils, eosinophil secretory ribonucleases, and eosinophil chemoattractants in host defense against PVM and related paramyxovirus pathogens in vivo.     

Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide.

It was previously reported that truncation or proteolytic removal of the C-terminal 16 amino acids (the R peptide) from the cytoplasmic tail of the murine leukemia virus (MuLV) envelope protein greatly increases its fusion activity. In this study, to investigate the specificity of the effect of the R peptide on the fusion activity of viral envelope proteins, we expressed simian immunodeficiency virus (SIV)-MuLV chimeric proteins in which the entire cytoplasmic tail of the SIV envelope protein was replaced by either the full-length MuLV cytoplasmic tail or a truncated MuLV cytoplasmic tail with the R peptide deleted. Extensive fusion of CD4-positive cells with the chimeric protein containing a truncated MuLV cytoplasmic tail was observed. In contrast, no cell fusion activity was found for the chimeric protein with a full-length MuLV cytoplasmic tail. We constructed another SIV-MuLV chimeric protein in which the MuLV R peptide was added to an SIV envelope protein cytoplasmic tail 17 amino acids from its membrane-spanning domain. No fusion activity was observed within this construct, while the corresponding truncated SIV envelope protein lacking the R peptide showed extensive fusion activity. No significant difference in the transport or surface expression was observed among the various SIV-MuLV chimeric proteins and the truncated SIV envelope protein. Our results thus demonstrate that the MuLV R peptide has profound inhibitory effects on virus-induced cell fusion, not only with MuLV but also in a distantly related retroviral envelope protein which utilizes a different receptor and fuses different cell types.     

Glycoprotein of nonpathogenic rabies viruses is a key determinant of human cell apoptosis

We showed that, unlike pathogenic rabies virus (RV) strain CVS, attenuated RV strain ERA triggers the caspase-dependent apoptosis of human cells. Furthermore, we observed that the induction of apoptosis is correlated with a particular virus antigen distribution: the overexpression of the viral G protein on the cell surface, with continuous localization on the cytoplasmic membrane, and large cytoplasmic inclusions of the N protein. To determine whether one of these two major RV proteins (G and N proteins) triggers apoptosis, we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to produce the G and N proteins of ERA and CVS individually. The induction of ERA G protein (G-ERA) expression but not of ERA N protein expression resulted in apoptosis, and G-ERA was more efficient at triggering apoptosis than was CVS G protein. To test whether other viral proteins participated in the induction of apoptosis, human cells were infected with recombinant RV in which the G protein gene from the attenuated strain had been replaced by its virulent strain counterpart (CVS). Only RV containing the G protein from the nonpathogenic RV strain was able to trigger the apoptosis of human cells. Thus, the ability of RV strains to induce apoptosis is largely determined by the viral G protein.     

Attenuated vesicular stomatitis viruses as vaccine vectors

We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.     

新城疫病毒F48E8株融合蛋白基因序列分析

本研究报道了新城疫病毒（ＮＤＶ）中国标准强毒株Ｆ４８Ｅ８融合蛋白（Ｆ）基因的序列。该基因核苷酸序列长度为１７００ｂｐ,编码由５５３个氨基酸组成的Ｆ０多肽。Ｆ０酶切激活部位序列为ＲＲＱＲＲ↓Ｆ,具有ＮＤＶ强毒的特征。Ｆ０中有３个主要由疏水性氨基酸组成的区域和６个可糖基化位点。经比较,ＮＤＶＦ４８Ｅ８株和Ｍｉｙａｄｅｒａ株、ＴｅｘａｓＧＢ株的氨基酸同源性分别为９３．６４％和９２．４１％。     

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.     

Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependent on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding.     

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.     

Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.     

The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.     

Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.

A cDNA copy of the RNA encoding the fusion (F) protein of Newcastle disease virus (NDV) strain Texas, a velogenic strain of NDV, was obtained and the sequence was determined. The 1,792-base-pair sequence encodes a protein of 553 amino acids which has essential features previously established for the F protein of virulent NDV strains. These include the presence of three strongly hydrophobic regions and pairs of dibasic amino acids in the pentapeptide Arg-Arg-Gln-Arg-Arg preceding the putative cleavage site. When inserted into a fowlpox virus vector, a glycosylated protein was expressed and presented on the surface of infected chicken embryo fibroblast cells. The F protein expressed by the recombinant fowlpox virus was cleaved into two polypeptides. When inoculated into susceptible birds by a variety of routes, an immunological response was induced. Ocular or oral administration of the recombinant fowlpox virus gave partial protection, whereas both intramuscular and wing-web routes of inoculation gave complete protection after a single inoculation.     

Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine.

We have mapped a linear epitope recognized by the virus-neutralizing monoclonal antibody 6-15C4 within the primary sequence of the G protein from the Evelyn-Rokitnicki-Abelseth strain of rabies virus. This was accomplished by using fragments of the rabies virus G protein and deduced amino acid sequences of neutralization-resistant variant rabies viruses. The monoclonal antibody 6-15C4 specifically recognized a synthetic peptide (peptide G5-24) which resembles the 6-15C4 epitope in structure. In addition, a tandem peptide constructed from the G5-24 peptide and a dominant TH cell epitope of the rabies virus N protein induced protective immunity against lethal rabies virus challenge infection in mice.     

Effects of the C-terminal truncation in NS1 protein of the 2009 pandemic H1N1 influenza virus on host gene expression

The 2009 pandemic H1N1 influenza virus encodes an NS1 protein with 11 amino acids (aa) truncation at the C-terminus. The C-terminal tail of influenza virus NS1 protein constitutes a nucleolar localization signal (NoLS) and is the binding domain of the cellular pre-mRNA processing protein, poly(A)-binding protein II (PABII). Here, our studies showed that the C-terminal-truncated NS1 of the 2009 pandemic virus was inefficient at blocking host gene expression, extension of the truncated NS1 to its full length increased the inhibition of host gene expression. Mechanistically, this increased inhibition of host gene expression by the full-length NS1 was not associated with nucleolar localization, but was due to the restoration of NS1's binding capacity to PABII. Furthermore, in vitro and in vivo characterization of two recombinant viruses encoding either the C-terminal 11-aa truncated or full-length NS1 of the 2009 pandemic virus showed that the C-terminal 11-aa truncation in NS1 did not significantly alter virus replication, but increased virus pathogenicity in mice.