- Volume 67, Issue 12, 1986

In eukaryotic cells, small nuclear and small cytoplasmic RNAs (sn- or scRNAs) are associated with distinct proteins, forming ribonucleoproteins (snRNPs or scRNPs). In the present study we analysed the protein composition as well as the small RNA pattern in non-infected and herpes simplex virus type 1 (HSV)-infected Vero cells. We found that concomitantly with the shut-off of host cell mRNA synthesis, synthesis of U-snRNAs was stopped. Due to their stability, however, U-snRNAs were still present in cells 36 h after HSV infection. Besides these RNAs, two novel small RNAs which we termed HVR1 and HVR2 were detected in infected cells. On the basis of their relative mobilities in urea gels, the apparent chain lengths of these newly synthesized RNAs were determined to be 255 and 154 nucleotides respectively. The small RNA-binding proteins Sm, RNP, Ro and La were found to increase up to 15-fold after HSV infection. The data presented suggest that new, virus-coded small RNPs are synthesized which might play a role in the maturation and regulation of HSV-coded RNA transcripts.

As an attempt to elucidate further the pathogenesis of human cytomegalovirus (HCMV) infection the replication of HCMV in primary human bone marrow cells (BMC) has been investigated. It was found that BMC held in culture in general were susceptible to HCMV infection. Compared to human embryonic lung cells, however, the replicative cycle of HCMV AD169 in BMC as determined by the analysis of viral protein and DNA synthesis was delayed and productive virus infection was restricted to a subset of BMC not exceeding 21% of the total cell population. Both of these phenomena may explain the short-term persistence of HCMV in BMC cultures which was observed over 3 months. By experiments with specifically enriched and depleted cell populations and by indirect double immunofluorescence experiments we found that both bone marrow fibroblasts and a subset of bone marrow stem cells supported productive virus infection. The finding that HCMV replicates in early stem cells of the human bone marrow may explain important aspects of the pathogenesis of HCMV infection including the presence of HCMV in peripheral blood leukocytes.

The susceptibility of human central nervous system cell lines to human cytomegalovirus (HCMV) and the fate of infected cultures were studied. Significant amounts of infectious progeny virus were produced in 118MGC glioma and IMR-32 neuroblastoma, but not in KGC oligodendroglioma cells when the cultures were infected with wild-type virus (HCMVwt) at an m.o.i. of 10 p.f.u. per cell. Further passage of infected 118MGC cells resulted in the establishment of a long-term persistent infection. This infection, designated 118MGC/Towne, continuously produced infectious virus (HCMVpi) with titres ranging from 102 to 105 p.f.u./106 cells up to 360 days post-infection (corresponding to 50 subcultures). Since no temperature-sensitive mutants, defective interfering particles or interferon-like activity were found in the 118MGC/Towne cultures, maintenance of the persistent infection seemed to be due to a balance between the release of infectious virus and the growth of uninfected cells. The HCMVpi produced in long-term persistently infected cultures was shown to be different from the HCMVwt originally used to infect by the following characteristics: (i) HCMVpi replicated slowly and yielded lower amounts of progeny virus than HCMVwt; (ii) HCMVpi induced a 73000 mol. wt. immediate early protein that was not synthesized in HCMVwt-infected cells; (iii) HCMVpi had a different DNA structure from that of HCMVwt. These results suggest that HCMVpi is a slower growing variant of HCMVwt and probably plays an important role in the maintenance of the persistent infection.

The relationship between serotype specificity and protein structure was studied by polyacrylamide gel electrophoresis, peptide mapping and radioimmune precipitation (RIP) of structural and non-structural proteins of the five U.S. serotypes of bluetongue virus (BTV). The surface proteins, VP2 and VP5, showed the most variation in size among the serotypes. Peptide mapping of the proteins showed that VP2 is unique for each of the U.S. serotypes. The nucleocapsid and non-structural proteins showed a high degree of conservation, whereas the other surface protein, VP5, showed intermediate conservation among the serotypes. Monospecific neutralizing antiserum produced in rabbits against each serotype was used in cross-RIP against cytoplasmic extracts prepared from cells infected with each BTV serotype. There were extensive cross-reactions among those proteins which showed a high degree of structural conservation, whereas VP2 was immunoprecipitated best in the homologous RIP system. Thus, a correlation between serotype specificity and protein structure was shown among the five U.S. serotypes of BTV.

Mice that did not contain antibodies to rotavirus were orally infected with murine rotavirus (EDIM strain) and observed over 7 days. As judged by ELISA, only the small intestine was infected, not the colon. The infection was biphasic, viral antigen peaks being observed at 48 h and approximately 120 h post-infection. Clinically evident diarrhoea was maximal at 72 h. Virus in the upper, middle and lower regions of the small intestine was mainly tissue-associated; most virus was found in the middle small intestine. Two peaks (48 h and 120 h post-infection) of virus antigen were observed in the colon, but these corresponded to luminal, not tissue-associated viral antigen. Only enterocytes in the upper two-thirds of villus epithelia were infected as judged by fluorescent-antibody analysis and transmission electron microscopy. Scanning electron microscopy revealed morphological appearances not hitherto correlated with the progress of the infection: villus tips were convoluted, corresponding to the shedding of virus-infected cells but the lower regions of infected villi were shrunken and considerably narrowed compared to tips.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus, New Jersey serotype, classified in complementation group E contain lesions in the NS gene, which manifest as marked electrophoretic mobility differences of the mutant NS proteins in SDS-polyacrylamide gels. We have cloned full-length cDNA copies of the mutant NS mRNAs, and have determined their nucleotide sequences. tsE1 and tsE3 had single nucleotide changes, and tsE2 had two nucleotide changes, compared to the wild-type NS gene. Three of the mutations were clustered in a region of 18 nucleotides. All the nucleotide differences resulted in amino acid substituations, which in each case changed the charge of the amino acid concerned. Analysis of the wild-type and mutant NS protein sequences by the method of Chou & Fasman indicated that single amino acid substitutions can radically alter the predicted secondary structure, and these data are discussed in relation to the observed electrophoretic mobility differences.

A rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to common and unique RNase T1 oligonucleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type- and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the laborious T1 oligonucleotide fingerprinting.

The antigenic relationships between Japanese encephalitis (JE) virus and several other flaviviruses have been investigated using anti-JE virus monoclonal antibodies (MAbs). Seventeen MAbs directed against envelope protein E of JE virus were characterized and divided into eight MAb groups based on reactivity patterns in haemagglutination inhibition test, neutralization (N) test, ELISA and competitive binding assay with JE virus. The results suggest the existence of at least eight epitopes on the E protein of JE viruses. Analysis of cross-reactivity of the antibodies with several other flaviviruses indicated these findings. (i) JE virus, belonging to West Nile (WN) subgroup, is antigenically closely related to viruses in the same subgroup, i.e. Murray Valley encephalitis (MVE), WN and St. Louis encephalitis (SLE) viruses. Of these three viruses, JE virus has the closest relationship with MVE virus. (ii) WN virus is relatively close to JE virus, whereas SLE virus is the least closely related. (iii) Dengue viruses types 1 and 2, which belong to another subgroup of flaviviruses, show markedly less antigenic homology to JE virus. (iv) One of the critical N sites on the E protein showed JE virus specificity. (v) Some cross-reactive antibodies which did not neutralize JE virus showed low but significant N activity against several other flaviviruses. Mixtures of several MAbs, which showed different reactivity patterns, potentiated the N activity against not only JE virus but also other members of the WN subgroup of flaviviruses, namely MVE, WN and SLE viruses.

In the present study, six lactate dehydrogenase-elevating virus (LDV) isolates obtained independently from inbred mice were compared by RNA oligonucleotide fingerprint analysis. The genome RNAs of four of the isolates gave unique fingerprint patterns. The patterns obtained for the other two isolates were similar, but not identical to one of the four unique patterns. These results indicate that more than one genotype of LDV exists and that virus isolates can be grouped by genotype. We have also demonstrated the presence of a 3′-terminal poly(A) tract by direct sequencing of 3′-32P-labelled LDV genome RNA. The presence of a 3′-terminal poly(A) tract distinguishes LDV from the members of the family Flaviviridae, which lack a 3′-poly(A), and justifies the current classification of LDV within the family Togaviridae.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

A measles virus (Hallé strain) cDNA library was prepared by cloning virus-induced mRNA directly into the expression vector PCD. Clones corresponding to the measles virus haemagglutinin (HA) gene were isolated and one, PCD-HA-15, which corresponded to the complete mRNA sequence, was further characterized. After transfection into COS-7 cells, measles virus HA antigen was detected by immunofluorescence. The [35S]methionine-labelled HA protein from transfected cells was immunoprecipitated by both polyclonal and monoclonal measles virus antibodies. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the PCD-HA-15 protein migrated in a manner identical to the virus-induced HA. Nucleotide sequence analysis established that the gene contained 1949 nucleotides [exclusive of poly(A)] and coded for a protein containing 617 amino acids. A single hydrophobic domain likely to represent the transmembrane region was identified at the N-terminus. A second overlapping reading frame coded for a protein containing 70 amino acids. This contained a short hydrophobic region (16 amino acids) and had two potential N-glycosylation sites. Comparison of the HA gene of the Hallé strain with the published sequence of the Edmonston strain showed that there was a high degree of conservation (99.3%).

The complete nucleotide sequence of the P+C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P+C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P+C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybridarrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P+C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Differences in the properties of homologous intracellular structural components of eight strains of subtype A and eight strains of subtype B of human respiratory syncytial (RS) virus were examined. The size of the fusion (F) protein cleavage products and the phosphoprotein (P) showed systematic differences between virus strains representing the two subtypes. The apparent mol. wt. in SDS-polyacrylamide gels under reducing conditions was 48000 (48K) and 46K to 47K for the cleavage product F1 in subtype A and B strains, respectively. The size of the F2 protein was 18K to 20K. The subtype B strains showed a slightly higher mol. wt. of this protein compared to the subtype A strains. The size of the P protein was 36K in subtype A strains, but only 34K in subtype B strains. Variations also occurred in the size of the glycoprotein (G) and the 22K to 24K structural protein. These variations did not correlate with the virus subtypes, but were strain-specific. The size of non-glycosylated forms of the F protein cleavage products was determined by use of material from tunicamycin-treated cells. A 44K to 45K non-glycosylated form of the F1 protein was detected with subtype A virus strains, but the corresponding protein of subtype B strains was not reproducibly identified, presumably due to instability in the absence of glycosylation or altered antigenicity. Monoclonal antibody immunosorbent-bound viral glycoproteins were partially digested with proteases. The pattern of breakdown products of the F1 protein was distinctly different between subtype A and B strains, but it was similar among strains of the same subtype. No subtype-specific pattern was seen in proteolytic digests of monoclonal antibody-bound G protein.

Intramuscular injection of mice with 2000 IU/g partially purified murine interferon (IFN) alpha/beta 3 h before the induction of IFN by intraperitoneally administered 3 µg/g poly rI:rC enhanced early IFN production. The differences between the serum IFN levels of IFN-pretreated and control animals were about 10-fold during the first 2 to 3 h of in vivo IFN production. In later stages these differences tended to decrease, and from 8 h post-induction they disappeared. IFN exerted its in vivo priming activity equally after intravenous, intraperitoneal or intramuscular injection. A similar enhancement of IFN production was observed when it was induced by poly rI:rC administered either intraperitoneally or intramuscularly. The duration of IFN pretreatment influenced the establishment of the in vivo primed state. Following administration of 2000 IU/g murine IFN alpha/beta intramuscularly, maximal priming developed after 3 h, and no primed IFN response was detected when the inoculation of IFN preceded inducer administration by 12 h or more. The manifestation of in vivo priming was optimal when 1500 to 3000 IU/g pretreating doses of IFN were applied. Reduction of the amount of injected IFN below this level markedly decreased priming, indicating a time- and dose-dependent induction of priming in vivo.

A panel of murine monoclonal antibodies (MAbs), selected by indirect ELISA, was prepared to an isolate (ACMV-JI) of the type strain of African cassava mosaic virus. Of the ten MAbs purified from ascitic fluids, four out of the five tested gave stronger reactions with ACMV-JI in double antibody sandwich ELISA (DAS-ELISA) than did rabbit polyclonal antibody. Six of the ten MAbs gave a precipitin reaction in immunodiffusion tests and nine trapped ACMV-JI particles in immunosorbent electron microscopy. Rabbit polyclonal antibody to ACMV reacted in DAS-ELISA not only with ACMV-JI and the Kenya coast strain of ACMV (ACMV-C) but also with five other geminiviruses known or suspected to have the whitefly Bemisia tabaci as a vector, though not with three geminiviruses that have leafhopper or unknown vectors. Of the ten MAbs studied in detail, four did not react with ACMV-C in indirect ELISA and only two reacted strongly. This supports other evidence that ACMV-C is a distinctive strain of ACMV and not merely a minor variant. The other whitefly-transmitted geminiviruses (bean golden mosaic, euphorbia mosaic, mung bean yellow mosaic and tomato golden mosaic viruses) and Australian tomato leaf curl virus reacted with two to five MAbs but each virus had a different pattern of reactivity. In contrast, solanum apical leaf curling virus, and the leafhopper-transmitted beet curly top and maize streak viruses, did not react with any of the MAbs. The results of competitive binding tests, when combined with the patterns of reaction of individual MAbs with different viruses, indicated that the MAbs were specific for nine distinct epitopes. Two MAbs reacted with all five whitefly-transmitted viruses, suggesting that the epitopes detected by these MAbs may be important for transmission by B. tabaci. Individual MAbs seem suitable for detecting and identifying ACMV, for distinguishing between ACMV-JI and ACMV-C, and for quantitative assays of other whitefly-transmitted geminiviruses.

When particles of rice dwarf virus were partially disrupted by treatment with EDTA, genome RNA was extruded. Electron microscope observations of these particles suggested that the genome RNA is packed within the virus particle as a supercoiled structure. Genome RNA, extracted using a newly devised two-step extraction method, appeared to consist of supercoiled segments of RNA complexed with protein.

Protoplasts from Nicotiana clevelandii were infected with velvet tobacco mottle virus (VTMoV) using inocula containing 2 µg/ml poly-l-ornithine (PLO), 8 µg/ml VTMoV and 0.02 m-potassium citrate pH 5.0. Typically about 47% of the inoculated protoplasts become infected. The amount of virus particles bound to inoculated protoplasts was increased by a decrease in pH, an increase in PLO concentration or an increase in virus concentration. The one-step growth curve of VTMoV in N. clevelandii protoplasts showed that, although large amounts of virus were synthesized, the multiplication was slower than that published for other viruses. Alpha-amanitin (50 µg/ml) had no effect on the rate of accumulation of virus coat protein or on the synthesis of either RNA species of VTMoV when added to cultures immediately after inoculation.

Treatment of particles of potato yellow dwarf virus, serotype SYDV, with non-ionic detergents solubilized two of the five structural proteins, G and M1. Since only these proteins were also recovered in lipid vesicles prepared either by a detergent dialysis technique or after organic extraction of purified virus using phosphatidylcholine in n-hexane, it is suggested that M2 protein is not membrane-associated but rather belongs to the core. The SYDV spike protein G was purified by isoelectric focusing during which it appeared to be heterogeneous in charge with most of the protein banding at pH 4.8. The same heterogeneity was observed after two-dimensional protein gel electrophoresis, where SYDV G protein was found largely at pH 4.8, whereas that of the other PYDV serotype, CYDV, was detected largely at pH 4.3. Antibodies raised against purified SYDV G protein reacted specifically with the G protein. When they were present during the inoculation step, G-specific antibodies were able to neutralize the infectivity of SYDV for insect vector cell monolayers, which suggests that G protein has a functional role in the inoculation process. Isolated SYDV G protein or SYDV envelope vesicles containing G protein inhibited the infection of vector cells by SYDV; however, only with SYDV envelope vesicles was the inhibition concentration-dependent. This indicated competition between virions and the vesicles during the inoculation process, possibly for recognition or attachment sites.

Infection of insect vector cells with the plant rhabdovirus potato yellow dwarf, serotype SYDV, was inhibited when the lysosomotropic substances chloroquine or ammonium chloride were present in the culture medium. Chloroquine inhibited infection totally at 1 mm, whereas inhibition by NH4Cl was maximum at 14 mm, where it was almost complete; lower concentrations of NH4Cl or, surprisingly, higher concentrations were less inhibitory. The number of cells infected at 25 mm-NH4Cl was the same as that infected in untreated controls. Binding of SYDV to the surfaces of cells was unimpaired by chloroquine. Sensitivity of SYDV infection to the lysosomotropic substances was observed only if the substances were administered during 150 min after inoculation. The inhibitory effect was abolished when the substances were removed from the medium. These results suggest that SYDV infection of the vector cells occurs by adsorptive endocytosis and has a lysosomal phase.