- Volume 91, Issue 5, 2010
Volume 91, Issue 5, 2010
- Animal
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- RNA viruses
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Hepatitis C virus superinfection of liver grafts: a detailed analysis of early exclusion of non-dominant virus strains
Liver transplantation (LT) of hepatitis C virus (HCV)-infected grafts into HCV-infected recipients leads to superinfection with two different virus strains. To characterize the virological outcomes of HCV superinfection immediately after LT, we performed phylogenetic analysis of a fragment of the NS5B gene in donor and recipient serum samples prospectively collected before and after LT, starting on day 1. In four of six cases, the donor strain finally prevailed, while in the remaining two cases, the native recipient strain overtook the donor quasispecies. Clonal sequence analysis showed that, in three cases, the expelled strain was undetectable 1 day after LT. Our study shows that superinfection with a different HCV strain can lead to the exclusion of one strain by the other as soon as the first day after LT. This would suggest that competition might not be limited to the replication level, but could also take place during virus entry.
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Cyclophilin A-independent recruitment of NS5A and NS5B into hepatitis C virus replication complexes
More LessThe mechanisms by which cyclophilin A (CypA) governs hepatitis C virus (HCV) replication remain unknown. Since CypA binds two essential components of the HCV replication complex (RC) – the polymerase NS5B and the phosphoprotein NS5A – we asked in this study whether CypA regulates their RC association. We found that CypA, via its isomerase pocket, locates in a protease-resistant compartment similar to that where HCV replicates. CypA association with this compartment is not mediated by HCV. Moreover, CypA depletion of RC does not influence NS5A and NS5B RC association, arguing against a model where CypA governs HCV replication by recruiting NS5A or NS5B into RC.
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Genotype 1 and global hepatitis C T-cell vaccines designed to optimize coverage of genetic diversity
Immunological control of hepatitis C virus (HCV) is possible and is probably mediated by host T-cell responses, but the genetic diversity of the virus poses a major challenge to vaccine development. We considered monovalent and polyvalent candidates for an HCV vaccine, including natural, consensus and synthetic ‘mosaic’ sequence cocktails. Mosaic vaccine reagents were designed using a computational approach first applied to and demonstrated experimentally for human immunodeficiency virus type 1 (HIV-Δ). Mosaic proteins resemble natural proteins, but are assembled from fragments of natural sequences via a genetic algorithm and optimized to maximize the coverage of potential T-cell epitopes (all 9-mers) found in natural sequences and to minimize the inclusion of rare 9-mers to avoid vaccine-specific responses. Genotype 1-specific and global vaccine cocktails were evaluated. Among vaccine candidates considered, polyvalent mosaic sequences provided the best coverage of both known and potential epitopes and had the fewest rare epitopes. A global vaccine based on conserved proteins across genotypes may be feasible, as a five-antigen mosaic cocktail provided 90, 77 and 70 % coverage of the Core, NS3 and NS4 proteins, respectively; protein coverage diminished with increased protein variability, dropping to 38 % for NS2. For the genotype 1-specific vaccine, the H77 prototype vaccine sequence matched only 50 % of the potential epitopes in the population, whilst a polyprotein three-antigen mosaic cocktail increased potential epitope coverage to 83 %. More than 75 % coverage of all HCV proteins was achieved with a three-antigen mosaic cocktail, suggesting that genotype-specific vaccines could also include the more variable proteins.
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RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem–loop structure, 5BSL3.2, and its negative strand
More LessThe hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem–loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem–loop structure of the negative strand in the replication process.
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Stable recombinants of bovine viral diarrhea virus containing a hepatitis C virus insert
More LessHere we report on a segment in the genomic 3′ non-translated region (3′NTR) of bovine viral diarrhea virus (BVDV) that is accessible for the insertion of foreign sequence elements such as the 5′NTR of hepatitis C virus. Recombinant viruses exhibited replication kinetics similar to those of the parental strain, and characterization of RNA species after several passages revealed that foreign inserts had the same genetic stability as the BVDV 3′NTR. The generation of such BVDV recombinants is relevant for several applications.
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RNA elements within the 5′ untranslated region of the West Nile virus genome are critical for RNA synthesis and virus replication
RNA elements within the flavivirus genome may play essential regulatory roles during virus replication. Here, recombinant West Nile virus (WNV) NS5 protein was used in combination with WNV subgenomic RNA templates to establish in vitro RNA-dependent RNA polymerase and RNA-binding assays. These assays identified mutations in the stem–loop A (SLA) region of the 5′ untranslated region (5′UTR) altering NS5 RNA synthesis and RNA-binding capability. These mutations were then introduced into the full-length WNV genome by reverse genetics. Further analysis of the mutant viruses showed that deletion of nt 46–60, which disrupted the stem and side loop of SLA, greatly compromised virus replication, whereas mutations that destroyed the top loop of SLA required for RNA synthesis in vitro did not significantly alter virus replication. These results suggest that SLA present in the 5′UTR of WNV is essential for RNA synthesis in vitro and for virus replication.
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Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection
More LessEukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E–eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.
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Recombination dynamics of human parechoviruses: investigation of type-specific differences in frequency and epidemiological correlates
Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20 % divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by beast in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.
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Variability in inhibition of host RNA synthesis by entero- and cardioviruses
More LessBoth entero- and cardioviruses have been shown to suppress host mRNA synthesis. Enteroviruses are also known to inhibit the activity of rRNA genes, whereas this ability of cardioviruses is under debate. This study reported that mengovirus (a cardiovirus) suppressed rRNA synthesis but less efficiently than poliovirus (an enterovirus). In contrast to poliovirus infection, the incorporation of BrUTP, fluorouridine and [14C]uridine in rRNA precursors was observed even during the late stages of mengovirus infection, although at a significantly reduced level. The cleavage of TATA-binding protein, considered to be one of the central events in poliovirus-induced transcription shutoff, was not detected in mengovirus-infected cells, indicating a difference in the mechanisms of host RNA synthesis inhibition caused by these viruses. The results also showed that functional leader protein is redundant for the suppression of host RNA synthesis by cardiovirus.
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Polypyrimidine tract-binding protein interacts with coxsackievirus B3 RNA and influences its translation
More LessWe have investigated the possible role of trans-acting factors interacting with the untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA. We show here that polypyrimidine tract-binding protein (PTB) binds specifically to both 5′ and 3′ UTRs, but with different affinity. We have demonstrated that PTB is a bona fide internal ribosome entry site (IRES) trans-acting factor (ITAF) for CVB3 RNA by characterizing the effect of partial silencing of PTB ex vivo in HeLa cells. Furthermore, IRES activity in BSC-1 cells, which are reported to have a very low level of endogenous PTB, was found to be significantly lower than that in HeLa cells. Additionally, we have mapped the putative contact points of PTB on the 5′ and 3′ UTRs by an RNA toe-printing assay. We have shown that the 3′ UTR is able to stimulate CVB3 IRES-mediated translation. Interestingly, a deletion of 15 nt at the 5′ end or 14 nt at the 3′ end of the CVB3 3′ UTR reduced the 3′ UTR-mediated enhancement of IRES activity ex vivo significantly, and a reduced interaction was shown with PTB. It appears that the PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA.
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Infection of in vivo differentiated human mast cells with hantaviruses
Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.
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Puumala hantavirus and Myodes glareolus in northern Europe: no evidence of co-divergence between genetic lineages of virus and host
More LessThe genus Hantavirus (family Bunyaviridae) includes negative-strand RNA viruses that are carried by persistently infected rodent and insectivore species. Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is a pathogenic hantavirus that causes outbreaks of mild haemorrhagic fever with renal syndrome across Europe. In northern Europe, PUUV is represented by several genetic lineages that are maintained by distinct phylogroups of bank voles. The present study describes sequences of new PUUV strains recovered from northern and southern regions of Scandinavia and compares phylogenetic relationships between north-European PUUV strains and M. glareolus. This analysis revealed contradictions in phylogenetic clustering and remarkable differences in estimated divergence times between the lineages of PUUV and its host, suggesting that the established PUUV lineages did not co-diverge with the distinct phylogroups of M. glareolus that carry them at present.
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Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia
Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2 %), Phlebotomus longicuspis (30.1 %), Phlebotomus papatasi (12 .0%), Phlebotomus perfiliewi (4.6 %), Phlebotomus langeroni (0.4 %) and Sergentomyia minuta (0.5 %). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.
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Role of host-specific amino acids in the pathogenicity of avian H5N1 influenza viruses in mice
Recent large-scale sequence analyses revealed ‘signature’ amino acids at specific positions in viral proteins that distinguish human influenza viruses from avian viruses. To determine the role of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses, we generated mutant viruses possessing signature amino acids in the PB2, PA and NP proteins of human influenza isolates (‘human-like amino acids’) in the genetic background of an avian H5N1 virus, and tested their pathogenicity in mice. We found that some of these mutants exhibited enhanced pathogenicity in mice, suggesting the involvement of these host lineage-specific amino acids in the pathogenicity of H5N1 avian influenza viruses in mammals.
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Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export
More LessThe influenza A virus RNA-dependent RNA polymerase produces capped and polyadenylated mRNAs in the nucleus of infected cells that resemble mature cellular mRNAs, but are made by very different mechanisms. Furthermore, only two of the 10 viral protein-coding mRNAs are spliced: most are intronless, while two contain unremoved introns. The mechanism(s) by which any of these mRNAs are exported from the nucleus is uncertain. To probe the involvement of the primary cellular mRNA export pathway, we treated cells with siRNAs against NXF1, Aly or UAP56, or with the drug 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II phosphorylation previously shown to inhibit nuclear export of cellular mRNA as well as influenza virus segment 7 mRNAs. Depletion of NXF1 or DRB treatment had similar effects, inhibiting the nuclear export of several of the viral mRNAs. However, differing degrees of sensitivity were seen, depending on the particular segment examined. Intronless HA mRNA and spliced M2 or unspliced M1 transcripts (all encoding late proteins) showed a strong requirement for NXF1, while intronless early gene mRNAs, especially NP mRNA, showed the least dependency. Depletion of Aly had little effect on viral mRNA export, but reduction of UAP56 levels strongly inhibited trafficking and/or translation of the M1, M2 and NS1 mRNAs. Synthesis of NS2 from the spliced segment 8 transcript was, however, resistant. We conclude that influenza A virus co-opts the main cellular mRNA export pathway for a subset of its mRNAs, including most but not all late gene transcripts.
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Pulmonary infection of mice with human metapneumovirus induces local cytotoxic T-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus
More LessHuman metapneumovirus (hMPV) is a major cause of upper and lower respiratory-tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory-tract infection with hMPV, we demonstrate the accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the airways and lungs at day 7 post-infection (p.i.), associated with cytotoxic T lymphocytes (CTLs) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th1 cytokines IFN-γ and interleukin (IL)-12 and antiviral cytokines (IFN-β), as well as chemokines Mip-1α, Mip-1β, Mig, IP-10 and CX3CL1. There was also a moderate increase in Th2-type cytokines IL-4 and IL-10 compared with uninfected mice. At 21 days p.i., a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response to the two related pathogens.
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The HR motif in the RNA-dependent RNA polymerase L protein of Chandipura virus is required for unconventional mRNA-capping activity
More LessChandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA : GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5′-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme–pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme–pRNA intermediate formation.
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Genomic and phylogenetic characterization of Merino Walk virus, a novel arenavirus isolated in South Africa
Merino Walk virus (MWV), a proposed novel tentative species of the family Arenaviridae, was isolated from a rodent, Myotomys unisulcatus, collected at Merino Walk, Eastern Cape, South Africa, in 1985. Full-length genomic sequence confirmed MWV as an arenavirus related distantly to Mobala, Mopeia and Ippy viruses, all members of the Old World arenavirus complex. We propose MWV as a tentative novel species in the Lassa–lymphocytic choriomeningitis virus complex, based on its isolation from a novel rodent species and its genetic and serological characteristics.
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Establishment and application of an infectious virus-like particle system for Marburg virus
More LessThe highly pathogenic Marburg virus (MARV) can only be investigated in high containment laboratories, which is time consuming and expensive. To investigate the MARV life cycle under normal laboratory conditions, an infectious virus-like particle (VLP) system was developed. The infectious VLP system is based on the T7-polymerase driven synthesis of a MARV-specific minigenome that encodes luciferase and is transcribed and replicated by the simultaneously expressed MARV nucleocapsid proteins NP, VP35, L and VP30. Transcription of the minigenome resulted in luciferase activity and replication resulted in encapsidated minigenomes. The encapsidated minigenomes, together with the viral matrix proteins VP40 and VP24 and the surface glycoprotein (GP), formed VLPs at the plasma membrane. Among the released pleomorphic VLPs, filamentous particles of 200–400 nm in length showed the highest capacity to induce reporter activity upon infection of target cells. To characterize the infectious VLP system, the intracellular concentration of one of the components was titrated, while all others were held constant. Intracellular concentrations of nucleocapsid proteins that resulted in highest replication and transcription activities also yielded VLPs with the highest ability to induce luciferase activity in target cells. High intracellular levels of VP40 maximized the release of VLPs, but reduced their ability to induce luciferase activity in target cells. The intracellular concentration of GP positively correlated with its incorporation into VLPs and their infectivity. Finally, we demonstrated that the infectious VLP system was suitable for rapid screening of neutralizing antibodies directed against MARV.
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Human immunodeficiency virus type 1 evasion of a neutralizing anti-V3 antibody involves acquisition of a potential glycosylation site in V2
It has been reported that the addition of a potential N-linked glycosylation site (PNGS) to the gp120 human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein provides protection against neutralizing antibodies (NAbs) by acting as a ‘glycan shield’. In this study, we induced insertion of a PNGS into the V2 region of HIV-1BaL with the KD-247 anti-V3 neutralizing monoclonal antibody. In the presence of KD-247 (200 μg ml−1) at passage five, viruses with 3 aa mutations in the C2 (T240S and I283T) and V3 (T319A) regions expanded from pre-existing variants. After six passages with KD-247 (>300 μg ml−1), a PNGS emerged in the V2 region in addition to C2 (T240S) and V3 mutations (R315K and F317L). A variant with a PNGS insertion in V2, but no V3 mutations was sensitive to KD-247, whereas a clone with a V2 PNGS insertion and mutations in V3 demonstrated a high level of resistance to KD-247. Replication kinetic analysis revealed that the F317L mutation in V3 played a compensatory role for fitness-loss caused by the PNGS insertion in V2. The evading HIV-1 variant did not revert back to the wild-type virus after 14 passages without KD-247. These findings demonstrate that the virus with fitness-loss mutations can replicate equally as well as the wild-type virus to acquire some key mutations in the V3 stem and the C2 region, and the compensated variants containing PNGS do not revert back to the ancestral virus even in the absence of NAb.
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Host factor pleiotrophin induces human immunodeficiency virus type 1 replication associated with inflammatory cytokine expression
More LessPleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding growth factors; it displays mitogenic activity for a wide variety of cells. In a previous study, we reported that PTN induces the stimulation of expression of inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-6, in quiescent human peripheral blood mononuclear cells (PBMCs) through B-lymphocyte binding. These results emphasize the importance of PTN in the regulation of inflammatory processes. Moreover, using in vitro infection of PBMCs or using PBMCs from AIDS patients, we showed that PTN was sufficient to induce human immunodeficiency virus type 1 (HIV-1) replication. Moreover, neutralization of TNF-α, IL-1β and IL-6 suppressed HIV replication in PTN-stimulated PBMCs. As these cytokines are potent upregulators of virus expression, these results should prove useful in investigating the role of PTN as a host factor in the regulation of pathological disorders in HIV-1 infection. Identification of this host factor could be important for understanding HIV disease and designating therapeutic approaches.
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Emergence of monoclonal antibody b12-resistant human immunodeficiency virus type 1 variants during natural infection in the absence of humoral or cellular immune pressure
Human immunodeficiency virus type 1 (HIV-1) resistance to broadly neutralizing antibodies such as b12, which targets the highly conserved CD4-binding site, raises a significant hurdle for the development of a neutralizing antibody-based vaccine. Here, 15 individuals were studied of whom seven developed b12-resistant viruses late in infection. The study investigated whether immune pressure may be involved in the selection of these viruses in vivo. Although four out of seven patients showed HIV-1-specific broadly neutralizing activity in serum, none of these patients had CD4-binding site-directed antibodies, indicating that strong humoral immunity is not a prerequisite for the outgrowth of b12-resistant viruses. In virus variants from one patient, who showed extremely weak heterologous and autologous neutralizing activity in serum, mutations were identified in the envelope that coincided with changes in b12 neutralization sensitivity. Lack of cytotoxic T-cell activity against epitopes with and without these mutations excluded a role for host cellular immunity in the selection of b12-resistant mutant viruses in this patient. However, b12 resistance correlated well with increased virus replication kinetics, indicating that selection for enhanced infectivity, possibly driven by the low availability of target cells in the later stages of disease, may coincide with increased resistance to CD4-binding site-directed agents, such as b12. These results showed that b12-resistant HIV-1 variants can emerge during the course of natural infection in the absence of both humoral and cellular immune pressure, suggestive of other mechanisms playing a role in the selective outgrowth of b12-resistant viruses.
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- DNA viruses
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Differential importance of highly conserved residues in UL24 for herpes simplex virus 1 replication in vivo and reactivation
More LessThe UL24 gene of herpes simplex virus 1 (HSV-1) is widely conserved among all subfamilies of the Herpesviridae. It is one of only four HSV-1 genes for which mutations have been mapped that confer a syncytial plaque phenotype. In a mouse model of infection, UL24-deficient viruses exhibit reduced titres, particularly in neurons, and an apparent defect in reactivation from latency. There are several highly conserved residues in UL24; however, their importance in the role of UL24 in vivo is unknown. In this study, we compared virus strains with substitution mutations corresponding to the PD-(D/E)XK endonuclease motif of UL24 (vUL24-E99A/K101A) or a substitution of another highly conserved residue (vUL24-G121A). Both mutant viruses cause the formation of syncytial plaques at 39 °C; however, we found that the viruses differed dramatically when tested in a mouse model of infection. vUL24-E99A/K101A exhibited titres in the eye that were 10-fold lower than those of the wild-type virus KOS, and titres in trigeminal ganglia (TG) that were more than 2 log10 lower. Clinical signs were barely detectable with vUL24-E99A/K101A. Furthermore, the percentage of TG from which virus reactivated was also significantly lower for this mutant than for KOS. In contrast, vUL24-G121A behaved similarly to the wild-type virus in mice. These results are consistent with the endonuclease motif being important for the role of UL24 in vivo and also imply that the UL24 temperature-dependent syncytial plaque phenotype can be separated genetically from several in vivo phenotypes.
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Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22
The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.
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Pseudorabies virus US3-mediated inhibition of apoptosis does not affect infectious virus production
More LessPreventing apoptosis during the early stages of infection of a host cell is generally thought to result in a higher yield of progeny virus. The US3 protein kinase of pseudorabies virus (PRV) and herpes simplex virus (HSV) is able to protect infected cells from apoptosis, which may be one of the reasons why both US3null PRV and US3null HSV replicate to lower virus titres in several cell types. However, such potential correlation between the higher amount of apoptosis in US3null virus-infected cells and the lower virus titres of US3null virus has not been investigated directly. In the current study, we found that a broad-spectrum caspase-inhibitor efficiently inhibited apoptosis in swine testicle and human laryngeal epidermoid carcinoma cells infected with US3null or wild-type (WT) PRV. However, inhibition of apoptosis did not affect US3null or WT PRV extracellular or cell-associated virus titres, nor did it restore the small plaque phenotype of US3null PRV.
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Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63
Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.
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Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex
In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.
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Dendritic cells are susceptible to infection with wild-type adenovirus, inducing a differentiation arrest in precursor cells and inducing a strong T-cell stimulation
More LessAdenovirus infection after stem cell transplantation is a significant cause of morbidity and mortality, especially in children. A robust T-cell response induced by dendritic cells (DC) is crucial for clearing the virus, suggesting their pivotal role for the response to human adenoviruses (HAdV). Despite the widespread use of adenoviral vectors, the properties and kinetics of HAdV infection of DC have not been addressed yet. We show that a recent clinical HAdV, subgenus C/serotype 2 (strain BB2000-61), infects cells of the myeloid lineage. Infected DC produce early and late viral antigens and show an altered expression of surface markers. Infection of monocytes renders them refractory to differentiation into DC. Additionally, HAdV-infected DC are strong stimulators of CD8+ T cells. In summary, HAdV seems to manipulate the immune response by infection of DC and possibly uses the infection of monocytes as a means to escape recognition by T cells.
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Human lung innate immune cytokine response to adenovirus type 7
Adenovirus (Ad) type 7 can cause severe infection, including pneumonia, in military recruits and children. The initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. Subsequently, monocytes become evident and, finally, there is a predominantly lymphocytic infiltrate. We have established that Ad7 infection of epithelial cells stimulates release of the neutrophil chemotaxin interleukin (IL)-8, and have extended these studies to a human lung tissue model. Here, we studied cytokine responses to Ad7 in human alveolar macrophages (HAM) and our human lung tissue model. Both ELISA and RNase-protection assay (RPA) data demonstrated that, upon Ad7 infection, IP-10 and MIP-1α/β are released from HAM. IP-10 and MIP-1α/β protein levels were induced 2- and 3-fold, respectively, in HAM 24 h after Ad7 infection. We then investigated induction of specific cytokines in human lung tissue by RPA and ELISA. The results showed that IL-8 and IL-6 were induced 8 h after infection and, by 24 h, levels of IL-8, IL-6, MIP-1α/β and MCP-1 were all increased. IP-10, a monocyte and lymphocyte chemokine, was also induced 30-fold, but only 24 h after infection. Immunohistochemistry staining confirmed that IL-8 was only released from the epithelial cells of lung slices and not from macrophages. IP-10 was secreted from both macrophages and epithelial cells. Moreover, full induction of IP-10 is likely to require participation and cooperation of both epithelial cells and macrophages in intact lung. Understanding the cytokine and chemokine induction during Ad7 infection may lead to novel ways to modulate the response to this pathogen.
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Evaluation of white spot syndrome virus variable DNA loci as molecular markers of virus spread at intermediate spatiotemporal scales
More LessVariable genomic loci have been employed in a number of molecular epidemiology studies of white spot syndrome virus (WSSV), but it is unknown which loci are suitable molecular markers for determining WSSV spread on different spatiotemporal scales. Although previous work suggests that multiple introductions of WSSV occurred in central Vietnam, it is largely uncertain how WSSV was introduced and subsequently spread. Here, we evaluate five variable WSSV DNA loci as markers of virus spread on an intermediate (i.e. regional) scale, and develop a detailed and statistically supported model for the spread of WSSV. The genotypes of 17 WSSV isolates from along the coast of Vietnam – nine of which were newly characterized in this study – were analysed to obtain sufficient samples on an intermediate scale and to allow statistical analysis. Only the ORF23/24 variable region is an appropriate marker on this scale, as geographically proximate isolates show similar deletion sizes. The ORF14/15 variable region and variable-number tandem repeat (VNTR) loci are not useful as markers on this scale. ORF14/15 may be suitable for studying larger spatiotemporal scales, whereas VNTR loci are probably suitable for smaller scales. For ORF23/24, there is a clear pattern in the spatial distribution of WSSV: the smallest genomic deletions are found in central Vietnam, and larger deletions are found in the south and the north. WSSV genomic deletions tend to increase over time with virus spread in cultured shrimp, and our data are therefore congruent with the hypothesis that WSSV was introduced in central Vietnam and then radiated out.
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Autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be substituted functionally by Rachiplusia ou multiple nucleopolyhedrovirus ODV-E56
More LessThe Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of virus host range. To assess the role of ODV-E56 in oral infectivity and host range, we constructed recombinant AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lacZ) in which ODV-E56 protein synthesis was eliminated by inserting a β-galactosidase (lacZ) expression cassette into the odv-e56 open reading frame. We also constructed a recombinant virus, Ac69GFP-Roe56, in which the native AcMNPV odv-e56 coding sequence was replaced with that of Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The odv-e56 recombinant viruses exhibited no alterations in polyhedron production and morphogenesis or in the production of infectious budded virus in cell culture. In bioassays using three lepidopteran host species, the oral infectivities of the odv-e56 mutant viruses Ac69GFP-e56lacZ and AcIEGFP-e56lacZ were profoundly impaired compared with those of wild-type and control recombinant viruses. Oral infectivity was restored fully by marker rescue of the odv-e56 mutant viruses with either the AcMNPV or the RoMNPV odv-e56 gene. In bioassays using two host species that are more susceptible to RoMNPV than to AcMNPV, Ac69GFP-Roe56 killed larvae with LC50 values similar to those of recombinant viruses expressing AcMNPV ODV-E56. This result indicated that replacement of the AcMNPV odv-e56 gene with the RoMNPV orthologue did not increase virulence against these two species.
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Analysis of cassava brown streak viruses reveals the presence of distinct virus species causing cassava brown streak disease in East Africa
More LessCassava brown streak virus (CBSV) isolates were analysed from symptomatic cassava collected between 1997 and 2008 in the major cultivation regions of East Africa. An analysis of complete RNA genomes of seven isolates from Kenya, Tanzania, Mozambique, Uganda and Malawi revealed a common genome structure, but the isolates clearly clustered in two distinct clades. The first comprised isolates from Kenya, Uganda, Malawi, north-western Tanzania and the CBSV described previously, and shared between 87 and 95 % nucleotide sequence identity, whilst the second included isolates from coastal regions of Mozambique and Tanzania, which shared only 70 % nucleotide sequence identities with isolates of the first clade. When the amino acid sequences of viral proteins were compared, identities as low as 47 % (Ham1) and 59 % (P1) between the two clades were found. An antiserum obtained against the capsid protein of a clade 1 isolate identified a 43 kDa protein in clade 1 isolates and a 45 kDa protein in clade 2 isolates. Several cassava cultivars were susceptible to isolates of clade 2 but resistant to those of clade 1. The differences observed both in biological behaviour and in genomic and protein sequences indicate that cassava brown streak disease in East Africa is caused by at least two distinct virus species. It is suggested that those of clade 1 retain the species name Cassava brown streak virus, whilst those of clade 2 be classified as Cassava brown streak Mozambique virus.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 100 (2019)
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Volume 99 (2018)
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Volume 98 (2017)
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Volume 97 (2016)
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Volume 96 (2015)
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Volume 93 (2012)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)