Extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus Ankara

Vaccinia virus is a structurally complex virus that multiplies in the cell cytoplasm. The assembly of Vaccinia virus particles and their egress from infected cells exploit cellular pathways. Most notably, intracellular mature viral particles are enwrapped by Golgi-derived or endosomal vesicles. These enveloped particles, enriched in virus-encoded proteins, migrate to the cell surface where they are released into the extracellular space through fusion of their outer envelope with the cell membrane. We report that baby hamster kidney cells productively infected with the modified vaccinia virus Ankara strain (MVA) also release extracellular vesicles containing virus-encoded envelope proteins but devoid of any virus cargo. Such vesicles were visualized on the cell surface by electron microscopy and immunogold labelling of the B5 envelope protein. A portion of the B5 protein was found to be associated with non-viral material in high speed ultracentrifugation pellets and displayed a buoyant density characteristic of exosomes released by some cell types. An unrelated transmembrane protein (CD40 ligand) encoded by the MVA genome was also incorporated into extracellular vesicles but not into the envelopes that surround extracellular enveloped virus. High speed pellets obtained by centrifugation of culture medium from cells infected with MVA encoding CD40 ligand displayed the ability to induce dendritic cell maturation suggesting that the ligand is on the outer surface of the extracellular vesicles. We propose that the formation of extracellular vesicles after vaccinia virus infection is a byproduct of the pathway leading to the formation of extracellular enveloped virus.     

Evidence for an alkaline protease in vaccinia virus.

Purified vaccinia virions (Lister strain) were found to contain a proteolytic activity that can use exogenous proteins as a substrate once the virions are disrupted at pH 10.6. Assays of proteolytic activity were carried out with labeled cellular proteins bound to an insoluble support. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of virus retained the activity. Temperature and pH dependence were observed as well as an inhibitory effect of heavy metal ions.     

Analyses of pea necrotic yellow dwarf virus-encoded proteins

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown—U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein–protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.     

Modulation of cellular machineries by Zika virus-encoded proteins

The strain of Zika virus (ZIKV) that circulated during the 2015 epidemic in Brazil has been associated with more than 2000 cases of microcephaly from September 2015 through November 2016. The viral genome determines the biology and pathogenesis of a virus and the virus employs its own gene products to evade host immune surveillance, manipulate cellular machineries, and establish efficient replication. Therefore, understanding the functions of virus-encoded protein not only aids the knowledge of ZIKV biology but also guides the development of anti-ZIKV drugs. In this review, we focus on 10 proteins encoded by ZIKV and summarize their functions in ZIKV replication and pathogenesis according to studies published in the past 6 years.     

Quantitative assay of recombinant vaccinia virus-encoded neomycin phosphotransferase in infected eukaryotic cell lysates

A method for the detection and quantitation of neomycin phosphotransferase (NPT II) activity in recombinant vaccinia virus (VV)-infected eukaryotic cell lysates is described. The assay is linear with respect to both protein concentration and time of incubation. Cytoplasmic extracts of cells infected with a recombinant VV expressing the bacterial neo gene exhibited NPT II levels more than 50-fold higher than those detected in extracts from either uninfected or VV-infected cells. These results indicate that interference from cellular or viral-induced ATPase activities is sufficiently low that NPT II enzyme activity can be measured in crude cell lysates without employing additional protein purification procedures.     

Interaction of vaccinia DNA-binding proteins with DNA in vitro.

The interaction between the components of the vaccinia DNA-protein complex isolated from infected cells has been studied. The rapid sedimentation of complex-associated DNA is destroyed by the addition of 2 M NaCl. However, the DNA is restored to a rapidly sedimenting state upon removal of the salt. This reconstitution is Pronase sensitive, indicating that conversion requires complex-associated protein. It was demonstrated that competitor DNA but not RNA from eucaryotic or bacterial sources inhibited restoration of rapid sedimentation of endogenous complex-associated DNA. The results suggest that complex-associated proteins responsible for DNA complexing are not specific for viral DNA.     

Immunodetection in vivo of beet necrotic yellow vein virus-encoded proteins

Open reading frames identified on the four genomic RNAs of beet necrotic yellow vein virus were cloned into bacterial expression vectors and resulting cl-fusion proteins expressed in Escherichia coli were used to raise polyclonal antibodies. This set of antisera was used to show the presence of 7 of 9 predicted viral proteins in mechanically inoculated Chenopodium quinoa leaves by the Western blot technique. Viral coat protein (p22) and its readthrough protein p85 encoded by RNA-2 could be detected in all subcellular fractions. Two other RNA-2-encoded proteins, p42 and p13, are predominantly associated with membranous structures. Another RNA-2-encoded protein, p14, as well as the two polypeptides p25 and p31, encoded by RNA-3 and -4, respectively, are soluble proteins. The viral proteins could first be detected about the time lesions became visible and increased thereafter except for p85, in which case the amount of the soluble form decreased with time. No protein could be detected corresponding to the RNA-1-encoded p237 protein or to the p15 species encoded by open reading frame V of RNA-2.     

Studies on the structural proteins of vaccinia virus. I. Structural proteins of virions and cores

Radioactively labeled vaccinia virions grown in L cells were dissociated to yield the constituent polypeptide chains, which were then subjected to electrophoresis in polyacrylamide gels. Mechanical fractionation of these gels yielded complex profiles in which at least 17 components, some major, some minor, could be reproducibly identified. The relative mass in each component has been calculated from the amount of radioactivity incorporated. The principal component (VSP-4) accounts for about 28% of the total viral protein mass. Cores derived from virions by chemical treatment contain the principal viral protein component, which is clearly multiple, as well as two minor ones (VSP-1 and VSP-2), which are probably single polypeptide species. These latter two components are the two slowest moving ones, and therefore most probably have the largest molecular weights. Treatment of virions with the nonionic detergent NP 40 liberates one major viral protein component (VSP-6). This component accounts for 18.7% of viral protein, the second highest amount, and is also multiple in nature.     

Expression of Mycobacterium tuberculosis and Mycobacterium leprae proteins by vaccinia virus.

Eight Mycobacterium tuberculosis and M. leprae genes were inserted into the vaccinia virus genome by in vivo recombination. The resulting virus recombinants were shown to express five different M. tuberculosis proteins (71, 65, 35, 19, and 12 kDa) and three M. leprae proteins (65 and 18 kDa and a biotin-binding protein) by Western immunoblot analysis, radioimmunoprecipitation, or black-plaque assay. When injected into BALB/c mice, the recombinants expressing the M. tuberculosis 71-, 65-, or 35-kDa protein and the M. leprae 65-kDa protein or the biotin-binding protein elicited antibodies against the appropriate M. tuberculosis or M. leprae protein. These vaccinia virus recombinants are being tested for the ability to elicit immune protection against M. tuberculosis or M. leprae challenge in animal model systems. The recombinants are also useful in generating target cells for assays aimed at elucidating the cellular immune responses to mycobacterial proteins in leprosy and tuberculosis. Furthermore, the M. tuberculosis 65-kDa protein and four of the other mycobacterial proteins share homology with known eucaryotic and procaryotic stress proteins, some of which may play a role in autoimmunity.     

Analysis of BoLA w6. Evidence for multiple subgroups

BoLA w6 is one of 16 specificities agreed upon at the 2nd International BoLA Workshop and many laboratories have produced sera reacting with this specificity. This paper presents evidence for at least four sub-groups in w6. Three Edinburgh sera showing identical reaction patterns in the 1st and 2nd BoLA workshops have been studied by absorption. One serum contained a single antibody population reacting with an epitope common to all w6 positive cells. The other two contained antibodies against the same common epitope, antibodies to epitopes on four subgroups and an antibody reacting only with one subgroup w6.1. Another Edinburgh serum contained two populations of antibodies. One reacting with the common epitope and one with an epitope on two or possibly three subgroups. Immunisation between w6 positive individuals produced antisera to two subgroups without producing antibodies to common epitopes. At least two additional subgroups are likely to exist. These results indicate the presence of specificities unique to individual subtypes coupled with epitopes common to some and all w6 subgroups.     

Herpes simplex virus-encoded ribonucleotide reductase: Evidence for the dissociation/ reassociation of the holoenzyme

³⁵S-labeled cells infected with herpes simplex virus type 1 (HSV-1), temperature-sensitive (ts) mutantts 1222 were used as a source of the large subunit of the viral ribonucleotide reductase (RR) to investigate the binding of the large (RR₁) and small (RR₂) subunits in the active enzyme. Mixing³⁵S-labeled RR₁ fromts 1222 with unlabeled RR₁/RR₂ complex from wild type (wt) infected cells resulted in the formation of a complex between³⁵S-labeled RR₁ and unlabeled RR₂, indicating that the complex between the RR₁ and RR₂ subunits is dynamic and subunit dissociation/reassociation occurs during enzyme function. Similar results were obtained when unlabeled HSV-2 RR was substituted for HSV-1 RR, demonstrating that the holoenzyme can be formed from the large subunit of HSV-1 RR and the small subunit of HSV-2.Requests for reprints should be addressed to Allan J. Darling, The Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, Scotland.     

Expression of hepatitis E virus putative structural proteins in recombinant vaccinia viruses.

Hepatitis E virus (HEV) is a polyadenylated, positive-stranded RNA virus which is a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries. The viral genome contains three different open reading frames (ORFs): ORF1, which is believed to encode nonstructural proteins, and ORF2 and ORF3, which are believed to encode structural proteins. The full-length putative structural proteins encoded by ORF2 and ORF3 of HEV have been cloned and expressed in recombinant vaccinia virus. Proteins encoded by ORF2 and ORF3 when expressed in vaccinia virus are recognized by pooled sera obtained from individuals with acute hepatitis E. Vaccinia-expressed viral gene products of HEV will have utility in characterizing the cell-mediated immune response to HEV.     

Studies on the structural proteins of vaccinia virus. II. Kinetics of the synthesis of individual groups of structural proteins

The time course of the synthesis of certain structural vaccinia virus proteins in mouse L fibroblasts was determined by pulse-labeling infected cells at intervals throughout the multiplication cycle and determining the degree of labeling of individual proteins of progeny virus after polyacrylamide gel electrophoresis. Overall viral structural protein synthesis reaches a peak in these cells at between 6 and 8 hours after infection, but then continues at a relatively high rate throughout the infection cycle. Different groups of viral structural proteins exhibit different patterns of synthesis. The synthesis of two proteins which are constituents of viral cores commences very early, as does that of a protein which is situated near the surface of the virion. These proteins are also synthesized in the presence of cytosine arabinoside, when viral DNA replication is inhibited, and are thus "early" proteins. The synthesis of the two early core proteins, but not that of the surface protein, appears to be subject to switch-off; however, switch-off is not absolute, and small amounts of these proteins continue to be synthesized throughout the infection cycle. The presence of structural viral proteins in "soluble" cell extracts (supernatant solutions after centrifuging at 20,000 g for 60 minutes) was also examined. The structural viral protein complement of such cell extracts bears only limited resemblance to that of virions. Some structural viral proteins appear to be synthesized in the form of polypeptide chains larger than those actually incorporated into virions; other viral proteins appear to aggregate rapidly after their synthesis. This is true particularly of those structural proteins which are the latest to be synthesized. The implications of these results are discussed.     

A map of the late proteins of vaccinia virus

SacI restriction fragments of vaccinia DNA were transferred to nitrocellulose filters and used to select for specific vaccinia RNAs by hybridization to RNA prepared from cells infected 8 hr previously with vaccinia virus. The selected RNAs were translated in vitro and the polypeptide products analyzed by SDS-PAGE. Each DNA restriction fragment gave rise to a different set of polypeptide bands. Approximately 60 bands were assigned positions on the SacI (SstI) map of vaccinia. Some bands comigrated with virion protein components and hence may represent structural proteins of vaccinia virus.     

Evidence that the major surface proteins of three Leishmania species are structurally related

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.     

Association of non-viral proteins with recombinant vaccinia virus virions

Summary Evidence is presented which suggests that recombinant vaccinia virus particles (VV : CAT), containing the bacterial chloramphenicol acetyl transferase gene, are capable of encapsidating both the foreign protein which they encode (CAT) as well as cellular enzymes such as thymidine kinase. These results are discussed with respect to using VV to passively introduce biologically-active proteins into cells or organisms.With 1 Figure     

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulse-chase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65^gag, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95^gag, or its precursor, Pr75^gag. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, $\text{35}\text{56}$, which is specific for the MuLV G_IX antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same $\text{35}\text{56}$ phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.     

Isolation of two DNA-binding proteins from the intracellular replication complex of vaccinia virus.

Two DNA-binding proteins, designated FP11 and FP14, have been purified from the viral replication complex which forms in the cytoplasm of HeLa cells during productive infection with vaccina virus. Polypeptide FP11 has an apparent molecular weight of 34,000 (by SDS-PAGE) and binds strongly to denatured DNA-cellulose columns, eluting as a narrow peak centered at 0.25 M NaCl. This polypeptide represents 30–40% of the [³H]lysine but only 17% of the [3H]methionine which can be incorporated into the complex. Polypeptide FP14 has an apparent molecular weight of 28,000 (by SDS-PAGE) and elutes from denatured DNA-cellulose as a relatively broad peak over the range 0.45–0.80 M NaCl. FP14 accounts for approximately 10% of incorporated radioactivity, regardless of whether lysine or methionine is employed as the radioactive precursor.     

Evidence for a multiple innervation of subcommissural ependymocytes in the rat.

Glandular ependymocytes of the rat subcommissural organ (SCO) receive a multiple innervation: a serotonergic afference identified by radioautography and at least one other input of a hitherto unknown transmitter content. Both types of terminals make well differentiated axo-glandular synapses with SCO-ependymocytes, but display different morphological characteristics. Serotonergic terminals being considered as inhibitory, it is suggested that the other type of terminal is excitatory.