Structural and nonstructural proteins of strain Colburn cytomegalovirus

The growth of most Rous sarcoma viruses (RSV) is severely restricted on MSB-1 cells (a line of chicken T lymphoblasts) in comparison to growth on chicken embryo fibroblast (CEF). Nonconditional transformation defective mutants of RSV from which the complete src region has been deleted (td RSV) are not subject to growth restriction. We examined the formation and integration of RSV and td RSV in MSB-1 cells following high multiplicity infection. Nearly equivalent quantities of the linear form of unintegrated RSV and td RSV DNA were formed in these cells during the first 10 hr after infection. Linear RSV DNA from MSB-1 cells could not be distinguished from linear RSV from CEF by restriction endonuclease analysis and by previously described transfection assays (P. E. Neiman, C. McMillin-Helsel, and G. M. Cooper, 1978, Virology 89,360–371). Beyond 10 hr after infection, and with progressive cell growth in the MSB-1 cultures, the level of RSV linear DNA rapidly decreased. Presumptive circular RSV DNA was detected only transiently, and at very low levels, about 15 hr after infection. Association of RSV DNA with high-molecular-weight chromosomal DNA, i.e., integration, was not detected in this study. In contrast, nearly constant levels of td RSV unintegrated linear DNA and, after 20 hr, circular DNA persisted in MSB-1 cells for at least 7 days after infection. Integration of td RSV proviral DNA was inefficient, occurring in only about 5% of MSB-1 cells (even at very high multiplicities of infection) in the first round of infection, and in 25–40% of cells by 3 days after infection. Almost all MSB-1 cells containing td RSV DNA produced virus. Analysis of eight nonconditional transformation defective mutants of RSV which retain the src region to different extents showed that all of these mutants replicated to the same normal titer on MSB-1 cells as on CEF without further deletion of the src region. Two temperature sensitive src mutants that thermal inactivation of the scr gene on MSB-1 cells at both 35° and 41°, indicating that thermal inactivation of the src gene product could not abrogate the replication block. These studies clearly demonstrate that the presence of the src region in RSV impedes the formation and/or integration of provirus in some types of host cells.     

Feline syncytium-forming virus proviral DNA Time of synthesis and relationship to the host cell genome

An infectious DNA assay has been used to study the time of synthesis of feline syncytium-forming virus (FSFV) proviral DNA, and also its relationship to the host cell genome. When extracted late in infection, infectious FSFV proviral DNA was associated with high molecular weight DNA with the same buoyant density as host cell DNA. Furthermore, the apparent size of this DNA could be reduced by shearing, suggesting that the fragments containing FSFV provirus also contained sequences not essential for viral replication. These data suggest that the DNA provirus of FSFV was integrated into chromosomal DNA late in infection. Under single-cycle growth conditions, infectious FSFV DNA was detected after 1 hr in the Hirt supernatant and after 2 hr in the Hirt pellet DNA. Progeny virus was not detected until at least 5 hr postinfection. It was concluded that the synthesis of free proviral DNA and its subsequent integration into the host cell genome may be essential events in the replication of foamy viruses.     

The synthesis and structure of visna virus DNA.

The synthesis of proviral DNA of visna virus was measured at various intervals after inoculation of sheep cell cultures at multiplicities of 0.3, 1, and 10 PFU/cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) molecular weight DNAs, and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb (1973). The appearance of infectious DNA in the Hirt supernatant (low molecular weight viral DNA) was biphasic at all multiplicities, apparently reflecting two rounds of synthesis of this DNA. The amount of infectious DNA in the Hirt precipitate fraction increased with time, reaching maximum levels in all cultures at the time of peak virus production. Hirt supernatant DNA consisted predominantly of molecules of molecular weight 6 × 10⁶, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate, in contrast, had a molecular weight of 166 x 106, suggesting its association with cellular sequences. The network test strongly suggested that the viral sequences were covalently linked with or integrated into the cellular DNA.     

Termperature-sensitive mutants of pseudorabies virus with differential effects on viral and host DNA synthesis

Mouse kidney cells infected with polyoma virus were labeled for 20 min with [5,6-³H]uridine late in infection. The rapidly-labeled RNA was extracted from whole cells and from the Hirt SDS/high salt supernatant fraction. The RNA was self-annealed and became resistant to ribonuclease digestion. The RNase-resistant RNA was isolated by chromatography on Sephadex G-100 and shown to be double-stranded RNA by several different criteria: resistance to RNase and to the combined effects of RNase and DNase, the characteristic buoyant density of double-stranded RNA in Cs₂SO₄, a buoyant density increase upon denaturation to that of single-stranded viral RNA marker, an inability to hybridize with complementary DNA unless denatured, and a high T_m value with a sharp transition to RNase sensitivity. Separated strands of the double-stranded RNA hybridized with high efficiency with component I of polyoma DNA. After self-annealing in formamide at low temperature, from 18 to 30% of the total rapidly-labeled viral RNA of infected cells sedimented at 11 S; 11 S corresponds in size to about 30% of the polyoma DNA. The extraction procedure for double-stranded viral RNA also yields double-stranded cellular RNA, but the double-stranded viral RNA can be further purified by using the Hirt supernatant, the RNaseresistance of the viral RNA coupled with its higher T_m, its greater sedimentation coefficient.These observations indicate that, late in infection of mouse cells, polyoma DNA is transcribed symmetrically over a considerable portion of its length, yielding self-complementary RNA that is distinguishable from the cellular self-complementary RNA.     

A physical map of the linear unintegrated DNA of Visna virus

Our previous studies have shown that during the lytic infection of sheep cells in culture, visna virus produces an unintegrated viral DNA which is a linear double-stranded molecule with a molecular weight of approximately 6 × 10⁶ daltons. We have now studied this DNA using the method of Southern to locate the cleavage sites of a number of restriction endonucleases on the unintegrated visna viral DNA. Using these sites, we have constructed a physical map of the linear viral DNA. In addition, our analysis has identified two species of closed circular viral DNA in the Hirt supernatant fraction of visna virus-infected cells.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

An adenovirus from Tupaia (tree shrew): growth of the virus, characterization of viral DNA, and transforming ability

An adenovirus was isolated from a kidney cell culture of an apparently healthy tree shrew (Tupaia belangeri) and termed Tupaia adenovirus (TAV). An extensive host range study with different animal and human cells revealed that only Tupaia cells were susceptible to TAV infection. Tupaia embryonic kidney cells are the cells of choice for the plaque assay and for the efficient production of cell-free TAV at a titer of 10⁸ PFU/ml within 36 hr post infection. TAV immortalizes skin fibroblasts of the New World monkey Callithrix jacchus. Purified TAV particles had a capsid diameter of 78–80 nm. The molecular weight of the viral DNA was 21.5 × 10⁶ as determined by contour length measurements. The buoyant density of TAV DNA was 1.706 g × ml^?1 as determined by isopycnic CsCl centrifugation. TAV DNA, when extracted with guanidinium hydrochloride, contains protein bound to its termini, since circles of double-stranded DNA were observed by electron microscopy, and restriction enzyme cleavage analysis of the TAV DNA-protein complex shows that the protein is associated with the two terminal TAV DNA fragments. The infectivity of the viral DNA-protein complex was 200-fold higher than TAV DNA. Cell biological and immunological data indicate that TAV is different from other known adenoviruses.     

The viral DNA replication complex of adenovirus 12

After infection of human embryo kidney cells in a resting state with adenovirus 12, viral DNA synthesis began at 24 hr post infection (p.i.) and reached its peak at 32 hr p.i. Both parental and nascent DNAs were present in the M-band fraction of nuclei of infected cells at 32 hr p.i. The DNA synthesizing activity in vitro with isolated nuclei and the M-band fractions began to increase at 24 hr p.i. and reached its maximum at 32 hr p.i. The DNA synthesized in vitro was viral as revealed by DNA-DNA hybridization. Electron microscopic autoradiograms of infected cells revealed that silver grains were found in association with bandlike inclusion bodies in the interior of the nucleus and did not associate with the nuclear membrane. After chase for 1 hr, most of the grains were found in the same state. These observations indicate that the viral DNA replication complex is formed in the interior of the nucleus and contained in the M-band fraction after fractionation.     

Effect of interferon on the exogenous Friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65^gag, and glycoprotein precursor, gPr85^env, to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65^gag, was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.     

Conversion of restrictive mouse cells to permissiveness during sequential and mixed double infection by murine leukemia viruses.

Conversion from restrictive (two-hit) to permissive (one-hit) kinetics was observed when N- or B-tropic murine leukemia viruses were titrated on mouse embryo fibroblasts of nonpermissive Fv-1 genotype that had previously been nonproductively infected with the same virus at an average multiplicity of one. The effect was transient, disappearing within about 24 hr after the first infection, and was abrogated by exposure of the first virus preparation to increasing doses of ultraviolet light, with a D₃₇ inactivation dose of 2550 ergs/mm², about three times that for inactivation of viral replication. Prior infection of nonpermissive mouse cells with the NZB xenotropic virus did not alter their restrictive response to later infection with ecotropic viruses. Low multiplicity infection of $\text{NIH}\text{3T3}$ cells with a B-tropic virus, followed by treatment with iodoeoxyuridine, failed to induce productive infection by endogenous or exogenous virus. Cells of F₁ hybrid Fv-1 genotype, which are restrictive for both N- and B-tropic viruses, were converted to permissiveness for virus of either tropism after low multiplicity infection with virus of the opposite tropism. No evidence of NB-tropic recombinant progeny could be detected under these experimental conditions. The implications of these experimental observations with respect to the mechanism of restriction governed by the Fv-1 gene are discussed.     

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.     

Unintegrated murine leukemia viral DNA in newly infected cells

Unintegrated virus-specific DNA has been detected by DNA-DNA hybridization in mouse embryo fibroblasts 3 hr postinfection with Rauscher leukemia virus. Control studies indicate that this DNA most probably represents newly synthesized rather than preinfection host viral sequences. The DNA is found primarily in two size categories. Using λ bacteriophage DNA fragments as markers the molecular weight of the larger size DNA has been estimated to be 2.25 × 10⁶–3.25 × 10⁶. The smaller-size DNA sediments slightly faster than the in vitro DNA product of the endogenous RNA-dependent polymerase reaction.     

Autographa californica multiple nucleopolyhedrovirus core gene ac92 (p33) is required for efficient budded virus production

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac92 is a core gene encoding a protein associated with occlusion derived virus (ODV), binds human P53 and also has flavin adenine dinucleotide linked sulfhydryl oxidase activity but its role in the virus life cycle is not known. To determine ac92 function a deletion virus (vAc^92KO) was generated and transfected Sf9 cells revealed that vAc^92KO infection was restricted primarily to single cells and budded virus (BV) titer was reduced over 99.99%. However, viral DNA replication was unaffected and development of occlusion bodies in vAc^92KO-transfected cells evidenced progression to very late phases of viral infection. AC92 localized to both the cytoplasm and nucleus, and was also associated with BV as well as ODV. In BV AC92 was detected in BV envelope and nucleocapsid fractions. Finally it was shown that the ac92 homologue from the Group II alphabaculovirus Mamestra configurata NPV maco96 could only partially rescue vAc^92KO.     

Adoptive Immunotherapy of Feline Immunodeficiency Virus with Autologous Ex Vivo-Stimulated Lymphoid Cells Modulates Virus and T-Cell Subsets in Blood

The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4⁺ counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection.     

Biological and biochemical studies on the inactivation of avian oncoviruses by ultraviolet irradiation.

The effects of ultraviolet (uv) irradiation on transforming and replicating capacities of avian oncoviruses and on the synthesis of virus specific products after infection with irradiated virus were studied. Different strains of nondefective avian sarcoma viruses were inactivated at the same rate following single-hit kinetics. The 37% survival dose D₃₇ (1/e) was 736 erg mm^?2 on average. A comparison of the inactivation kinetics in a focus assay (transforming capacity) and an infectious center assay (replicating and transforming capacity) showed no partial inactivation of the virus genome; focus and infectious center formation were inactivated at the same rate. Similar results were obtained when the replicating capacity of the avian sarcoma virus was measured in a plaque assay; focus and plaque formation were inactivated at the same rate. No repair of the uv damage by either complementation or recombination with exogenous or endogenous avian leukosis virus could be demonstrated. The rates of inactivation of avian sarcoma virus assayed in focus and infectious center tests on chick embryo fibroblasts expressing or not expressing chicken helper factor, on chick embryo cells preinfected with RAV-1, and on Peking duck cells were identical. Nondefective avian sarcoma virus and deletion mutants of avian sarcoma virus defective for replication or transformation were inactivated at the same rate. Biochemical analysis of the DNA extracted from a Japanese quail tumor cell line (QT-6) 26 hr after infection with irradiated avian sarcoma virus strain B77 showed a decrease of total virus specific DNA and of full-length covalently closed circular (form I) viral DNA synthesis with increase of the uv dose. Virus-specific RNA synthesis, measured by hybridization of labeled RNA extracted from chicken embryo fibroblasts infected with irradiated virus to viral DNA, and particle production, assayed by uridine incorporation, were also inhibited with increasing uv dose. The inactivation rates for virus-specific DNA and RNA synthesis and for particle production were very similar, but lower than the rate for the loss of infectivity.     

Synthesis of the precursor to avian RNA tumor virus internal structural proteins early after infection.

The synthesis of the 76,000-dalton precursor (Pr 76) of the avian RNA tumor virus internal structural proteins was studied as a function of time after infection of chick embryo fibroblasts (CEF) with avian myeloblastosis virus (AMV). During the course of infection, cells were pulse-labeled with [³⁵S]methionine, lysed, and the labeled viral polypeptide precursor was precipitated with antibody against detergent-lysed AMV. Pr 76 was detected by SDS gel electrophoresis of immune precipitates.The earliest time at which Pr 76 synthesis could be detected was 3 hr after infection. Pr 76 synthesis remained low (about 1% of the level of synthesis several days after infection) and constant from 3 until 7 hr after infection. Between 7 and 9 hr after infection, Pr 76 synthesis increased by fivefold.Cells treated with cycloheximide during the first 8 or 12 hr after infection showed an 85–90% inhibition of Pr 76 synthesis and virus production measured late during infection. This finding does not necessarily imply that early viral protein synthesis is required for a productive infection, because cycloheximide also inhibited chick embryo fibroblast DNA synthesis.If cells were treated prior to and during infection with actinomycin D or cytosine arabinoside, precursor synthesis was still observed early (3 to 7 hr after infection). The amount of precursor synthesized early in the presence of inhibitors was similar to that synthesized in the absence of inhibitors, suggesting that the incoming RNA served as a messenger RNA for Pr 76.     

Recombinant chicken interferon-alpha inhibits the replication of exogenous avian leukosis virus (ALV) in DF-1 cells

Chickeninterferon alpha (ChIFNα) belongs to type I IFNs that are important antiviral cytokines. We investigated whether ChIFNα plays a role in avian leukosis virus (ALV) infections of chickens. Firstly, we explored the immune response to ALV in vivo by measuring cytokine expression profiles in the spleens and bursas of chickens during the late stages of ALV-J infection. The results indicated that ALV-J infection could induce a mixed Th1/Th2 cytokine response by elevating levels of both interleukin-2 (IL-2) and IL-10. In contrast, tumor necrosis factor alpha (TNF-α) levels decreased in the spleen while interferon beta (IFNβ) and Toll-like receptor 7 (TLR7) expression levels in the bursa increased significantly. This indicated that ALV-J stimulates a Type I IFN response. Next, we found that different ALV subgroups or strains up-regulated chicken IFN regulatory factor 3 (ChIRF-3) promoter activity, suggesting that ALV infection could trigger Type I IFNs pathway in vitro. Accordingly, we further investigated ChIFNα antiviral effects on ALV replication in DF-1 cells by successfully expressing recombinant ChIFNα in Escherichia coli (E. coli) strain BL21. The specific activity of the purified rChIFNα protein was determined to be 4 × 10⁷ U/mL. When added at 4000 U/mL, the recombinant protein restrained ALV replication as measured by decreases in viral protein p27 levels and mRNA expression. This new reagent may be useful for prophylactic and therapeutic drug design.