R.Walter Schlesinger

Identification and characterization of type 2 dengue virus replicative intermediate and replicative form RNAs

Abstract KB cells infected with dengue type 2 virus synthesize three types of RNA with sedimentation coefficients greater than 18 S. The first is soluble in 2 M LiCl, sediments as a discrete species at about 20 S, and is greater than 95% RNase resistant. The second is a discrete 40 S species, insoluble in 2 M LiCl, and more than 95% sensitive to digestion by RNase. The third, also insoluble in 2 M LiCl, is “heterogeneous” in size, sedimenting between 20 and 28 S, and is 50–70% RNase resistant. Pulse-chase studies demonstrate the “heterogeneous” and 20 S RNAs to be intermediate in the synthesis of the virus 40 S genomic RNA. The data from agarose-formaldehyde gel analysis of these RNAs are in agreement with our identification of the “heterogeneous” and 20 S RNAs as the replicative intermediate (RI) and replicative form (RF) RNAs, respectively.

Sialic acid contents of sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties.

Abstract Sindbis virus grown in Aedes albopictus cells, in contrast to virus grown in vertebrate cells, lacks sialic acid. The presence or absence of sialic acid has no effect on the antigenic properties of these viruses as tested by complement fixation, hemagglutination inhibition, neutralization, and immune virolysis. The particle/hemagglutination unit and particle/plaque-forming unit ratios are not significantly different for virus grown in CEF (chick embryo fibroblast), BHK, or A. albopictus cells.

An arginine-dependent step in the maturation of type 2 adenovirus

Abstract Of all amino acids in Eagles basal medium, only arginine is essential for synthesis of complete, infectious progeny virus in KB cells infected with type 2 adenovirus. Omission of any one or all of the other AAs from the medium permits limited virus replication, but none whatever can be detected in absence of medium arginine. When arginine was restored to infected cultures after 28–32 hours of deprivation, infectious virus was made without detectable lag and increased exponentially at the “normal” rate. Simultaneous addition of FUDR failed to inhibit virus production, suggesting that viral DNA had been synthesized in absence of arginine. Accordingly, when thymidine-3H was added to cultures only during arginine starvation, label was recovered in virus produced following restoration. Immunofluorescence and hemagglutination tests showed that “early” antigen as well as proteins associated with the hexon and penton-fiber subunits were synthesized during the arginine-free period. Thus all previously identified components of the mature virion appear to be made, at least to a limited extent, in the absence of external arginine. When the arginine restored to previously deprived infected cultures was tritium-labeled, radioactivity was associated with the purified virus yield. It is proposed that arginine plays a specific role in a late synthetic step essential for virion maturation.

Studies on the nature of dengue viruses: V. Structure and development of dengue virus in vero cells

Abstract Purified dengue-2 virus was examined by negative staining. The particles were round and averaged about 50 nm in diameter. Treatment of virus with 4.0 M NaCl resulted in the appearance of “cores” 26 nm in diameter. No definitive pattern of symmetry could be discerned. Dengue-2 virus was grown in Vero cells and the kinetics of virus release was shown to be similar to that described previously in KB cells. At 18 hr after infection the Vero cells showed marked cytoplasmic vacuolization, whereas the later stages (24–36 hr) were characterized by accumulation of particles in crystalloid arrays which were surrounded by membranes. Evidence is presented that these crystalloid aggregates were composed of mature virions. While dengue-2 virus predominantly accumulated in cytoplasmic vacuoles, Sinbdis virus in Vero cells matured at the cellular plasma membrane. Thus Sindbis virus and dengue-2 virus, a group A and group B arbovirus, respectively, appear to differ with respect to their morphogenesis in Vero cells.

Adenoviruses: The Nature of the Virion and of Controlling Factors in Productive or Abortive Infection and Tumorigenesis*

Publisher Summary The chapter discusses the various properties of the adenoviruses, such as the nature of virion and the controlling factors in the productive or abortive infection. The dissection of the virion and its component parts, summarized in this chapter, has provided essential markers for the exploration and correlation of these biological variables, the mapping of phage chromosomes, based on deletions in conditional lethal mutants, required the structural and functional identification of phage precursors and subunits, such that each identifiable moiety of the adenovirion will help in relating biological activity to structural and molecular properties of the viral genomes. For a full understanding of the biology of adenovirus infection, as of other virus-cell systems, it is important to recognize that uniformity and synchrony of response is a deliberately created experimental artifact resulting only from high multiplicity infection of competent cells. This type of response may be absent in natural infections, which does not occur under conditions of low multiplicity infection, and may be fundamentally irrelevant in adenoviral tumorigenesis and abortive infection. For the sake of simplicity, any infection with complete adenovirus particles not leading to production of infectious progeny will be defined as “abortive.” The term “complete” in this context, implies that the same virions can induce productive infection in some suitable indicator cell. Hence, these viruses can be characterized, operationally, as host dependent conditionally lethal, either the permissive host cell supplies some function needed for virus replication that the non permissive cell lacks, or the latter imposes a restriction not present in the permissive one. Both alternatives could, in a purely descriptive way, satisfy the need for working hypotheses to explain the various examples of abortive infection with adenoviruses.

Studies on the nature of dengue viruses: I. Correlation of particle density, infectivity, and RNA content of type 2 virus☆

Type 2 dengue virus was harvested from KB cell cultures. The infected culture medium was extracted with fluorocarbon, and virus particles were concentrated and purified by two cycles of centrifugation on layers of fluorocarbon (density 1.574 g/ml). Equilibrium density centrifugation in CsCl revealed two major hemagglutinating components of average densities 1.19 and 1.24. The 1.24 component contained 99% of the residual plaque-forming activity and occasionally could be further fractionated into two subcomponents. Partially purified, P32-labeled viral HA fractions of different densities were adsorbed on goose erythrocytes, and the virus-RBC complexes were subjected to chemical fractionation. The amounts of label associated with RNA decreased from the densest to the least dense samples and were directly proportional to the PFU titers. Most of the non-RNA label was accounted for in the phospholipid fraction.

Studies on the nature of dengue viruses: II. Characterization of viral RNA and effects of inhibitors of RNA synthesis☆

Abstract The plaque-forming titer of 32 P- or 3 H-uridine-labeled type 2 dengue virus is proportional to the amount of label incorporated into a homogeneous species of RNA of sedimentation coefficient 45 S, estimated molecular weight 3.3 × 10 6 . The average base composition (per 100 nucleotides) is A = 30.7, G = 26.4, C = 21.3, U = 21.6. Lack of base pairing and degradation by pancreatic RNase indicate that it is a single-stranded molecule. In addition, another kind of RNA, with a different base composition than the 45 S RNA and a sedimentation coefficient of 6–8 S, is found. This RNA is associated with the HA band which moves more slowly in a linear sucrose gradient than does the major infectious band. The origin and significance of this RNA are unknown. Actinomycin D permits undiminished or slightly increased virus synthesis. 5-Fluorouridine or 6-azauridine inhibits infectious virus production in KB cells. Inhibition experiments with 6-azauridine, with or without reversal by uridine, show that RNA synthesis essential for virus maturation begins 6 hours after infection and precedes release of mature virions by 6–7 hours. Inhibition of HA formation (or release) by 6-azauridine follows the same dose response and time course as that of infectious virus.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation: I. Clonal stability of three biological characteristics

Abstract After several hundred generations in vitro, HT cells originally derived from type 12 adenovirus (Ad.12)-induced hamster tumors have retained their malignant character, the capacity to synthesize Ad.12-specific antigen(s), and a significantly reduced capacity, compared with that of normal hamster cells, to produce infectious type 2 adenovirus (Ad.2) in response to superinfection. Of four independently derived cell lines studied, one (HT2) consistently produces less Ad.2 than the other three lines. No evidence has been obtained for “rescue” of Ad.12 among the yields from Ad.2-superinfected HT cells. The low yield from such cells is not due to poor adsorption of inoculum virus. Thirteen clones of two cell lines (HT2 and HT8) have been established after microdrop isolation of single cells. The fact that all clones tested have retained the three characteristics suggests genetic homogeneity of the parental populations. In particular, the differing extent to which synthesis of Ad.2 is depressed in the two parental lines is reflected in the response of their derived clones. The evidence argues against the idea that HT cell populations consist of a majority of genetically stable “resistant” and a minority of stably “susceptible” cells.

Oligomeric organization and strain-specific proteolytic modification of the virion M2 protein of influenza A H1N1 viruses

The M2 protein of influenza A H1N1 strains PR8, WS, and WSN is present in homooligomeric forms in virions grown in the allantoic cavity of embryonated eggs. The bulk of the virion M2 is detected as tetramers and dimers. The oligomeric forms of PR8 virions differ from those of WS and WSN not only in apparent molecular weight (MW) but also in that they seem to be composed of two types of monomers differing in MW by approximately 1.5 kDa. Evidence from monoclonal antibody binding (or lack of it) and from in vitro trypsin digestion suggests that, in ovo, the external NH2 region of some PR8 M2 monomers is proteolytically trimmed, resulting in heterogeneous oligomers composed of cleaved and uncleaved monomers in different proportions.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation: II. Nature of the restricted response to type 2 adenovirus

Abstract The response of cultured cells derived from type 12 adenovirus-induced hamster tumors (HT cells) to superinfection with type 2 adenovirus (Ad.2) has been studied in detail. The low yield of infectious Ad.2 from such cultures has been shown, by infectious center and mass yield analyses, to be due to the presence of a small number of virus-producing cells. However, all superinfected HT cells are capable of producing viral structural antigen(s), provided the input multiplicity of Ad.2 is at least of the order of 10–100 PFU per cell. The restricted production of infectious progeny is not mediated by a transmissible interfering factor such as interferon. HT cells are as competent to produce vesicular stomatitis virus as are normal hamster cells. SV40-transformed hamster cells and normal hamster cells produce comparable (large) amounts of type 2 adenovirus. The evidence is consistent with the hypothesis that the response of HT cells to superinfection with Ad.2 is controlled by persistent genetic material of type 12 adenovirus responsible for the initiation of the malignant change. Several considerations are discussed which suggest that the response to superinfection is a more specific indicator of genome persistence than other identified properties of HT cells and that, operationally, the relation of the Ad.12 genome to HT cells may be analogous to that of a defective prophage to its bacterial host.