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PubMed2004

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating.

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.

Adenosine Triphosphate↗
PubMed2004

Influence of polypeptide size and intracellular sorting on the induction of epitope-specific CTL responses by DNA vaccines in a mouse model.

We have analysed the influence of size, intracellular localisation, and sorting of various human immunodeficiency virus type 1 (HIV-1)-derived Gag and Env polypeptides containing well defined H2(d)-restricted cytotoxic T lymphocyte (CTL) epitopes on the induction of a humoral and cellular immune response after DNA vaccination. Thus, expression vectors were generated based on RNA- and codon-optimised genes encoding (i). budding competent full-length Gag, (ii). a myristylation defect mutant GagMyr(-), (iii). the isolated p24 capsid moiety of Gag as well as variants of these proteins, which were C-terminally fused HIV gp120-derived V3 epitope (R10I), respectively. These constructs were compared to different minitopes each encoding one of the H2(d)-restricted Gag epitopes A9I and E10F or the V3 epitope R10I that were directly linked to the C-terminus of an Ad2-E3 protein-derived ER signal peptide. Immunological evaluation of these constructs in BALB/c mice revealed that both, the budding competent as well as the intracellular Gag proteins were-irrespective of their molecular weights-equally efficient in the priming of Gag-specific humoral and cellular immune responses. In addition, the capacity of these constructs to stimulate Gag-specific humoral as well as H2-K(d) and H2-L(d) restricted cellular immune responses was not influenced by C-terminal fusion of the immunodominant H2-D(d) restricted V3 epitope. Chimeric GagV3 polyproteins encoding all three major CTL epitopes within a continuous polyprotein were more efficient to stimulate epitope-specific cellular immune responses than the selected minitopes. In addition, the minitopes failed to induce epitope-specific antibody responses. These results clearly show the advantages of complex polypeptides over minitopes regarding the induction of strong humoral and cellular immune responses.

AIDS Vaccines↗
PubMed2005

Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine.

Listeria monocytogenes is an attractive biologic vaccine vector against HIV because it induces a strong cell mediated immune response, can be delivered by mucosal routes, can be readily manipulated to express viral antigens, and is easy and inexpensive to produce. Proof of concept studies have been performed using HIV Gag expressing recombinant L. monocytogenes in the mouse. Here we report the development and validation of recombinant L. monocytogenes to be evaluated in the FIV/cat model of HIV. Using a simplified approach to introduce individual and polyprotein FIV gag genes, we show that recombinant L. monocytogenes containing the entire gag expresses the full-length Gag polyprotein in a soluble secreted form. A DNA vaccine plasmid (pND14-Lc-env) that replicates in Gram positive bacteria and contains the FIV SU (gp100) and the ectodomain of TM (gp40) in a eukaryotic expression cassette was transfected into LM-gag to create LM-gag/pND14-Lc-env. After infection of target cells with LM-gag/pND14-Lc-env in vitro, both FIV Gag and Env proteins were detected in soluble cell lysates. Whether previous exposure to L. monocytogenes affects the immunogenicity of LM-gag/pND14-Lc-env was determined in cats infected with wild-type L. monocytogenes orally and/or subcutaneously. After a single oral dose of LM-gag/pND14-Lc-env, cats with existing anti-L. monocytogenes immune responses developed anti-FIV Gag IgA titers in vaginal secretions, saliva, and feces. Similarly, FIV Gag and Env specific IFN-gamma ELISPOT responses were measurable in spleen and lymph node but at a statistically higher frequency in cats exposed to a single subcutaneous dose of wild-type L. monocytogenes versus cats exposed both subcutaneously and orally. The FIV/cat model will provide a useful challenge system to determine whether recombinant L. monocytogenes can protect against a lentivirus in its natural host after challenge by the routes common to HIV transmission.

Animals↗
PubMed2004

Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo.

Heterosigma akashiwo (Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family (Marnaviridae), related to picorna-like viruses.

Amino Acid Sequence↗
PubMed2004

Generation and analysis of an RNA vaccine that protects against coxsackievirus B3 challenge.

Coxsackievirus B3 (CVB3) is an important human pathogen that causes substantial morbidity and mortality but, to date, no vaccine is available. We have generated an RNA-based vaccine against CVB3 and have evaluated it in the murine model of infection. The vaccine was designed to allow production of the viral polyprotein, which should be cleaved to generate most of the viral proteins in their mature form; but infectious virus should not be produced. In vitro translation studies indicated that the mutant polyprotein was efficiently translated and was processed as expected. The mutant RNA was not amplified in transfected cells, and infectious particles were not produced. Furthermore, the candidate RNA vaccine appeared safe in vivo, causing no detectable pathology following injection. Finally, despite failing to induce detectable neutralizing antibodies, the candidate RNA vaccine conferred substantial protection against virus challenge, either with an attenuated recombinant CVB3, or with the highly pathogenic wt virus.

Animals↗
PubMed2005

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication.

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.

Amino Acid Sequence↗
PubMed2006

An internal ribosome entry site promotes translation of a novel SIV Pr55(Gag) isoform.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

Animals↗
PubMed2004

Evidence for a polytopic form of the E1 envelope glycoprotein of Hepatitis C virus.

The polyprotein precursor of the Hepatitis C virus (HCV) contains multiple membrane-spanning domains that define the membrane topology and subsequent maturation of the viral structural proteins. In order to examine the biogenesis of the E1-E2 heterodimeric complex, we inserted an affinity tag (S-peptide) at specific locations within the envelope glycoproteins. In particular, and based on the prediction that the E1 glycoprotein may be able to assume a polytopic topology containing two membrane-spanning domains, we inserted the affinity tag within a putative cytoplasmic loop of the E1 glycoprotein. The HCV structural polyprotein containing this tag (at amino acids 295/296) was highly expressed and able to form a properly processed and noncovalently associated E1-E2 complex. This complex was bound by murine and conformation-dependent human monoclonal antibodies (MAbs) comparably to the native untagged complex. In addition, MAb recognition was retained upon reconstituting the tagged E1-E2 complex in lipid membrane as topologically constrained proteoliposomes. Our findings are consistent with the model of a topologically flexible E1 glycoprotein that is able to adopt a polytopic form. This form of the E1-E2 complex may be important in the HCV life cycle and in pathogenesis.

Amino Acid Sequence↗
PubMed2005

A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) is essential for virus growth in a murine macrophage-like cell line.

The DA subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein, which is translated out of frame with the polyprotein from an alternative AUG, 13 nucleotides downstream from the authentic polyprotein AUG. By a 'loss of function' experiment using a mutant virus, DAL*-1, in which the L* AUG is mutated to an ACG, L* protein is shown to play an important role in virus persistence, TMEV-induced demyelination, and virus growth in macrophages. In the present study, we established an L* protein-expressed macrophage-like cell line and confirmed the importance of L* protein in virus growth in this cell line.

Animals↗
PubMed2005

Translational control by viral proteinases.

Most RNA viruses have evolved strategies to regulate cellular translation in order to promote preferential expression of the viral genome. Positive strand RNA viruses express large portions, or all of their proteome via translation of large polyproteins that are processed by embedded viral proteinases or host proteinases. Several of these viral proteinases are known to interact with host proteins, particularly with the host translation machinery, and thus, encompass the dual functions of processing of viral polyproteins and exerting translation control. Picornaviruses are perhaps the best characterized in regards to interaction of their proteinases with the host translation machinery and will be emphasized here. However, new findings have shown that similar paradigms exist in other viral systems which will be discussed.

Animals↗
PubMed2006

A virus between families: nucleotide sequence and evolution of Strawberry latent ringspot virus.

Several clones of golden ginger mint (Mentha x gracilis, 'Variegata') were found infected with Strawberry latent ringspot virus (SLRSV). The virus was purified and cloned and the complete nucleotide sequence of a mint isolate was obtained. RNA 1 consists of 7,496 nucleotides excluding the poly-A tail and encodes a polyprotein with signature enzymatic motifs found in other picorna-like plant viruses. RNA 2 consists of 3,842 nucleotides excluding the poly-A tail, encoding a polyprotein that is processed to a putative movement protein and the two coat proteins of the virus. A satellite RNA of 1,117 nucleotides was associated with this isolate encoding for a putative protein of 31 kDa. Phylogenetic analysis revealed that SLRSV shares characteristics with members of the Cheravirus, Fabavirus, Comovirus and Sadwavirus genera indicative of the uniqueness of SLRSV. The close relationship of SLRSV with these genera led to the examination of aphid and beetle transmission of the virus with, however, negative results.

Animals↗
PubMed2003

Antiviral inhibition of the HIV-1 capsid protein.

During the assembly stage of the human immunodeficiency virus (HIV) replication cycle, several thousand copies of the viral Gag polyprotein associate at the cell membrane and bud to form an immature, non-infectious virion. Gag is subsequently cleaved by the protease, which liberates the capsid proteins for assembly into the polyprotein shell of the central core particle (or capsid) of the mature virus. Viral infectivity is critically dependent on capsid formation and stability, making the capsid protein a potentially attractive antiviral target. We have identified compounds that bind to an apical site on the N-terminal domain of the HIV-1 capsid protein and inhibit capsid assembly in vitro. One compound, N-(3-chloro-4-methylphenyl)-N'-[2-[([5-[(dimethylamino)-methyl]-2-furyl]-methyl)-sulfanyl]ethyl]urea) (CAP-1), is well tolerated in cell cultures, enabling in vivo antiviral and mechanistic studies. CAP-1 inhibits HIV-1 infectivity in a dose-dependent manner, but does not interfere with viral entry, reverse transcription, integration, proteolytic processing, or virus production, indicating a novel antiviral mechanism. Significantly, virus particles generated in the presence of CAP-1 exhibit heterogeneous sizes and abnormal core morphologies, consistent with inhibited CA-CA interactions during virus assembly and maturation. These findings lay the groundwork for the development of assembly inhibitors as a new class of therapeutic agents for the treatment of AIDS.

Anti-HIV Agents↗
PubMed2004

Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Amino Acid Motifs↗
PubMed2001

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells.

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.

Animals↗
PubMed2001

Nucleotide sequence of black currant reversion associated nepovirus RNA1.

The RNA1 of black currant reversion associated nepovirus (BRAV) is 7711 nucleotides (nt) long, excluding the poly-A tail, and contains one long open reading frame (ORF) which is translated into a polyprotein of 2094 amino acids. The 5' NTR of BRAV RNA1 is 66 nt long and 78% identical with RNA2 5' NTR only over the first 57 nucleotides. The 3' non-translated region (3'NTR) is 1360 nucleotides long, and after the first 24 nucleotides 95% identical with the 3'NTR of RNA2. RNA1 3'NTR contains several stretches, 694-24 nucleotides in length, which are 60-80% similar to corresponding areas of the other viruses of the subgroup c of nepoviruses (BLMV, CLRV, PRMV or TomRSV). The 2094 amino acids-long polypeptide encoded by BRAV RNA1 is 33% identical with that of PRMV between amino acids 9 and 2057, and has significant similarity also to those of other nepoviruses and comoviruses. Conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cysteine protease and the RdRp core domains, known to occur in the polyproteins of different viruses of the picornavirus-like supergroup, are all detected in the amino acid sequences encoded by BRAV RNA1.

3' Untranslated Regions↗
PubMed2003

Encephalomyocarditis virus (EMCV) proteins 2A and 3BCD localize to nuclei and inhibit cellular mRNA transcription but not rRNA transcription.

We have followed the viral processing cascade and polyprotein precursor fates during encephalomyocarditis virus (EMCV) infection of HeLa cells using a panel of monoclonal antibodies (mAbs). Within the first 2-4 h of infection, signals of antibodies specific for the 2A, 3B(VPg), 3C(pro) and 3D(pol) proteins were found to co-localize in nucleoli at the rRNA synthesis and cellular protein B23 (nucleophosmin) sites. Cellular fractionation identified viral protein precursor 3BCD as the common source of the P3-region antibody signals. Previously thought to be a minor product of the polymerase region cleavage pathways, the nuclear targeting of this precursor was localized with engineered mutations to five P2 and P3 region polyprotein processing sites. A nuclear localization motif (NLS), similar to that in many yeast ribosomal proteins, was identified near the N-terminus of the 3D(pol) sequence. Point mutations within this motif prevented nuclear and nucleolar localization by all forms of 3B(VPg), 3C(pro) and 3D(pol), and were lethal to the virus because they also prevented genome replication. However, viral RNA synthesis was not required for nucleolar transport and 3BCD was found in nuclei, even when the 3D(pol) was inactivated. Co-immunoprecipitation experiments showed a tight association between 3BCD and B23 (nucleophosmin), suggesting a possible ribosomal protein-like mechanism for nuclear transport. Infected cell extracts analyzed with microarrays, quantitative slot-blots and pulse-labeling experiments confirmed a nearly complete shutoff of host pol-II-dependent mRNA synthesis during EMCV infection, in reactions that depended on wild-type 2A protein. In contrast to human rhinovirus-16 infection, rRNA synthesis by pol-I and pol-III were not turned off by EMCV, although the cellular concentration of rRNA decreased during infection, relative to control samples. The data suggest that nuclear targeting by 2A and 3BCD may be responsible for regulating cellular mRNA and rRNA transcription during infection, perhaps via a proteolytic mechanism catalyzed by the endogenous 3C(pro) sequence.

3C Viral Proteases↗
PubMed1997

Identification of bovine viral diarrhea virus nonstructural polypeptide NS4B/P38.

The bovine pestivirus polyprotein is processed into at least 11 mature polypeptides. Previous studies with polyprotein region-specific antiserum raised against beta-galactosidase fusion proteins or synthetic peptides allowed the assignment of viral non-structural proteins to specific segments of the BVDV genome. However, the gene product from the NS4B/P38 region of the viral genome remained to be demonstrated directly. BVDV cDNA fragments predicted to encode part of NS4B/P38 (from codon 2521 to 2838 of the BVDV open reading frame) and a portion of NS5A/P58 (from codon 3008 to 3340) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Polyclonal rabbit antibodies prepared against each of the purified fusion proteins, GST-2521-2838 and GST-3008-3340, were used to immunoprecipitate viral polypeptides present in BVDV-infected cell lysates. Rabbit antiserum to GST-2521-2838 bound a polypeptide of 38 kDa identified as the mature NS4B/P38 polypeptide; while anti GST-3008-3340 lacked this specificity and bound NS5A/P58. Moreover, both antisera recognized a 96 kDa polypeptide, previously identified as a NS4B-NS5A/PP96 precursor. The function of the newly identified and highly conserved NS4B/P38 protein in viral replication remains to be determined.

Animals↗
PubMed1998

Expression of the human immunodeficiency virus gag gene products by a replication-incompetent herpes simplex virus vector.

A recombinant replication-incompetent herpes simplex virus vector (vd120/Gag) that expressed the human immunodeficiency virus 1 gag gene and part of the pol gene that encodes the HIV-1 protease was constructed. Examination of cells infected with vd120/Gag revealed the presence of the Gag polyprotein Pr55gag by 12 h post-infection, as well as abundant levels of the proteolytically processed 24-kDa capsid protein. Analysis of vector-infected cells and culture supernatant indicated that the majority of the 24-kDa protein remained cell-associated. Although the HIV-1 Gag polyprotein was produced in vd120/Gag-infected cells, there was no evidence of HIV virus-like particle production upon examination of vector-infected cells by transmission electron microscopy.

Animals↗