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Biomedical subjects

P Palese

Publications and source records attributed to P Palese.

At least 55 records · Page 3Linked to original sources
PubMed1996

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

Animals↗
PubMed1996

Characterization of in vivo primary and secondary CD8+ T cell responses induced by recombinant influenza and vaccinia viruses.

We characterized the in vivo primary and secondary murine CD8+ T cell responses induced by immunization with influenza and vaccinia viruses, which were engineered to express the same H-2K(k)- and H-2K(d)-restricted epitopes. Our results show that the induction and magnitude of the primary CD8+ T cell response closely depends on the viral dose used for immunization, while it is not affected by the route of immunization. The induction of secondary CD8+ T cell responses appears to be highly restricted, as suggested by the lack of in vivo expansion of antigen-specific CD8+ T cells after repeated immunization with the same virus. In contrast, a 20- to 30-fold increase in the frequency of antigen-specific CD8+ T cells could be induced after combined immunization with recombinant influenza and vaccinia viruses. These findings may provide the basis for the development of new prophylactic and therapeutic strategies to prevent or control intracellular infections and certain malignancies.

Animals↗
PubMed1996

Nonconserved nucleotides at the 3' and 5' ends of an influenza A virus RNA play an important role in viral RNA replication.

The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form "panhandle" or "fork" structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5' end or of the nucleotides between position 15 and the start codon at the 3' end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3' and the 5' ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of infuenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3' and 5' ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment.

Animals↗
PubMed1996

A plasmid-based reverse genetics system for influenza A virus.

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.

Base Sequence↗
PubMed1996

Interaction cloning of NS1-I, a human protein that binds to the nonstructural NS1 proteins of influenza A and B viruses.

The yeast interaction trap system was used to identify, NS1-I (for NS1 interactor), which is a human protein that binds to the nonstructural NS1 protein of the influenza A virus. NS1-I is a human homolog of the porcine 17beta-estradiol dehydrogenase precursor protein, to which it is 84% identical. We detected only one NS1-I mRNA species, of about 3.0 kb, in HeLa cells, and the NS1-I cDNA was found to have a coding capacity for a 79.6-kDa protein. However, immunoblot analysis detected predominantly a 55-kDa protein in human cells, suggesting that NS1-I, like the porcine 17beta-estradiol dehydrogenase, is posttranslationally processed. Using an in vitro coprecipitation assay, we showed that NS1-I interacts with NS1 proteins from extracts of cells infected with five different influenza A virus strains as well as with the NS1 of an influenza B virus. The fact that influenza A and influenza B virus NS1 proteins bind to NS1-I suggests that this cellular protein plays a role in the influenza virus life cycle.

Amino Acid Sequence↗
PubMed1995

Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import.

We have fluorescently labeled one of the eight genomic segments of influenza virus RNA and a recombinant influenza viral protein, the nucleoprotein (NP), to investigate the requirement for their uptake into nuclei of digitonin-permeabilized cells. We found that the influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein. Thus, at 0 degrees C it docks at the nuclear envelope only in the presence of the heterodimeric karyopherin (either karyopherin alpha 1 beta or karyopherin alpha 2 beta), and docking is competitively inhibited by an unlabeled NLS containing substrate. Like other NLS-containing proteins, at 20 degrees C NP is imported into the nucleus after further addition of the GTPase Ran and of p10. In contrast, the fluorescently labeled, 890-nucleotide-long viral RNA segment does not dock to the nuclear envelope or enter the nucleus either in the presence of exogenous cytosol or of karyopherin heterodimer, Ran, and p10. However, in the presence of NP the RNA is able to dock and enter the nucleus with transport requirements indistinguishable from those for docking and entry of NP. These data indicate that uptake of the influenza virus RNA segment is not via a signal in the RNA but via an NLS of a viral protein such as NP.

Animals↗
PubMed1995

NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein.

We used the yeast interactive trap system to identify a cellular protein which interacts with the nucleoprotein of influenza A viruses. This protein, nucleoprotein interactor 1 (NPI-1) is the human homolog of the yeast protein SRP1. SRP1 was previously identified as a suppressor of temperature-sensitive RNA polymerase I mutations (R. Yano, M. Oakes, M. Yamaghishi, J. Dodd, and M. Nomura, Mol. Cell. Biol. 12, 5640-5651, 1992). A full-length cDNA clone of NPI-1 was generated from HeLa cell poly A + RNA. The viral nucleoprotein, which had been partially purified from influenza A/PR/8/34 virus-infected embryonated eggs, could be coprecipitated from solution by glutathione agarose beads complexed with a bacterially expressed glutathione-S-transferase-NPI-1 fusion protein, confirming the results of the yeast genetic system. Antisera raised against NPI-1 identified a 60-kDa polypeptide from total cellular extracts of both HeLa and MDBK cells. The viral nucleoprotein was coimmunoprecipitated from influenza A/WSN/33 virus-infected MDBK cells by anti-NPI-1 sera, demonstrating an interaction of these two proteins in infected cells. Similarly, NPI-1 was coimmunoprecipitated from MDBK cells by anti-NP sera. These experiments suggest that NPI-1 plays a role during influenza virus replication.

Amino Acid Sequence↗
PubMed1995

Presentation by a major histocompatibility complex class I molecule of nucleoprotein peptide expressed in two different genes of an influenza virus transfectant.

Major histocompatibility (MHC) class I glycoproteins are specialized to present to CD8+ T cells, peptides that originate from proteins synthesized within the cytoplasm. Conventional killed vaccines are unable to get into the cell cytoplasm and therefore fail to expand the CD8+ T cell population. We have created a novel influenza transfectant virus, R10, which carries an immunogenic peptide from the nucleoprotein (NP) of PR8 influenza virus in its hemagglutinin (HA) and another similar peptide in its HK influenza virus NP. The two peptides are both presented by H-2Db and bind with approximately equal affinity. They can compete with one another for binding to H-2Db. Yet in cells infected with R10, both peptides are presented efficiently enough to expand the respective cytotoxic T lymphocyte (CTL) precursors in vivo and to serve as targets for CTL lysis in vitro. It has been proposed that proteins bearing signal sequences may be processed by a transporter-independent pathway. To investigate this, we infected the transporter-deficient cell line RMA-S with the R10 virus to see if the NP peptide expressed by the HA would be presented. The result shows that even the presence of a signal peptide in the HA does not overcome the lack of a transporter function, suggesting that the presentation of both peptides is dependent on functional transporter proteins. Our data also suggest the feasibility of creating by genetic engineering, recombinant vaccines expressing multiple epitopes that can effectively stimulate a cellular immune response.

Amino Acid Sequence↗
PubMed1995

Influenza virus vectors.

The establishment of reverse genetics methods to manipulate influenza virus genomes has allowed the generation of recombinant (transfectant) influenza viruses expressing foreign sequences. Strategies for the construction of influenza virus vectors include the insertion of foreign epitopes into influenza virus glycoproteins, the expression of polyproteins, and the rescue of bicistronic genes into infectious viruses. Influenza virus vectors have been obtained which express both B- and T-cell epitopes from different pathogens. These constructs have been shown to mount in immunized animals systemic and local antibody responses, and/or cytotoxic T-cell responses against the expressed epitope. The available evidence suggests that influenza virus vectors may be attractive candidates for the development of effective vaccines against different diseases.

Amino Acid Sequence↗
PubMed1995

The cytoplasmic tail of the neuraminidase protein of influenza A virus does not play an important role in the packaging of this protein into viral envelopes.

We have rescued a transfectant influenza virus, NA/TAIL(-), whose neuraminidase (NA) protein lacks the predicted cytoplasmic tail. The virus was attenuated (one log10 reduction) both in tissue culture and in mouse lungs. Attenuation correlated with a 50% reduction of the level of NA in infected cells and levels of incorporation of the tail-less NA protein into viral particles paralleled that in infected cells. This result indicates that the signal for packaging of the NA protein into the viral envelope is not located in its cytoplasmic domain.

Animals↗
PubMed1995

Measles virus phosphoprotein (P) requires the NH2- and COOH-terminal domains for interactions with the nucleoprotein (N) but only the COOH terminus for interactions with itself.

A mammalian two-hybrid system was used to characterize protein-protein interactions between the measles virus nucleoprotein (N) and phosphoprotein (P). Progressive deletions at both the amino- and carboxy-termini of P facilitated the mapping of two distinct domains on P that are important for interaction with N: (i) a domain mapping predominantly within the C-terminal 100 amino acids and (ii) a domain composed of the extreme amino-terminal residues. Using the same two-hybrid assay, we discovered that the P protein interacts strongly with itself. In contrast to the N-P interaction, only a single C-proximal domain of P was essential for P-P interaction.

Amino Acids↗
PubMed1995

Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus.

Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.

AIDS Vaccines↗
PubMed1995

Influenza B viruses with site-specific mutations introduced into the HA gene.

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA. Next, a full-length copy of the HA gene from influenza B/Maryland/59 virus was cloned. Following transfection of this RNA, we rescued transfectant influenza B viruses which contain a point mutation introduced into the original cDNA. A series of mutants which bear deletions or changes in the 5' noncoding region of the influenza B/Maryland/59 virus HA gene were constructed. We were able to rescue viruses which contained deletions of 10 or 33 nucleotides at the 5' noncoding region of the HA gene. The viability of these viruses implies that this region of the genome is flexible in sequence and length.

Amino Acid Sequence↗
PubMed1995

Mutations within noncoding terminal sequences of model RNAs of Sendai virus: influence on reporter gene expression.

A reverse-genetics system employing the chloramphenicol acetyltransferase (CAT) reporter gene has been established previously for Sendai virus. We utilized PCR-directed mutagenesis to introduce nucleotide additions, deletions, and/or substitutions within terminal Sendai virus RNA sequences. The influence of these mutations on replication-transcription of these model Sendai-CAT RNAs was analyzed by assaying CAT activity. Results from these experiments indicate that (i) Sendai-CAT RNAs expressing wild-type levels of CAT activity conform to the Sendai virus rule of six, (ii) apparent exceptions to the rule of six exist in that the 5' terminus of the Sendai-CAT RNA is more tolerant than the 3' terminus of nucleotide additions or deletions, and (iii) the 3' leader region of Sendai-CAT RNA appears to be sensitive not only to mutagenesis (single-nucleotide addition or deletion) but also to changes in its total nucleotide length.

Base Sequence↗
PubMed1994

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1. The second ORF encodes the NA protein; its translation is achieved via an internal ribosomal entry site which is derived from the 5' noncoding region of the human immunoglobulin heavy-chain-binding protein mRNA. The GP2 (79 amino acids) and HGP2 (91 amino acids) polypeptides are expressed in cells infected with the corresponding transfectant virus. The HGP2 polypeptide, which contains transmembrane and cytoplasmic domains identical to those of the hemagglutinin (HA) protein of influenza A/WSN/33 virus, is packaged into virus particles. This novel influenza virus system involving an internal ribosomal entry site element may afford a way to express a variety of foreign genes in mammalian cells.

Animals↗
PubMed1994

Characterization of the polyadenylation signal of influenza virus RNA.

It has been shown that a stretch of uridines (U's) near the 5' end of the virion RNA of influenza A virus is the polyadenylation site for viral mRNA synthesis. In addition, the RNA duplex made up the 3' and 5' terminal sequences adjacent to the U stretch is also involved in polyadenylation. We have further characterized the polyadenylation signal of influenza virus RNA with a ribonucleoprotein transfection system. We found that the optimal length of the U stretch is 5 to 7 uridine residues. We also showed that the upstream sequence at the 5' end is not involved in polyadenylation and that the optimal distance between the 5' end and the U stretch is 16 nucleotides. The combination of these features defines the polyadenylation site and differentiates this signal from other U stretches scattered throughout the genomes of influenza viruses.

Base Sequence↗
PubMed1994

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

Amino Acid Sequence↗