The expression of viral functions is necessary for recombination of a herpesvirus (pseudorabies)

To determine whether viral functions are necessary for recombination of the pseudorabies virus genome in infected cells, we have used as a model system marker rescue at the permissive temperature (PT) and nonpermissive temperature (NPT) of a temperature sensitive mutant (tsG1) deficient in the immediate-early (180K) protein. Two restriction fragments, both of which can rescue tsG1 at the PT but only one of which encompasses the whole immediate-early gene and can complement tsG1, were compared for their ability to rescue the mutant at the NPT. Although both restriction fragments rescued the mutant with equal frequency at the PT, only the fragment which could express the immediate-early 180K protein prior to recombination, i.e. could complement tsG1, rescued the mutant at the NPT. We conclude that the expression of viral functions is necessary for high frequency recombination of the pseudorabies virus genome.     

Specific complex of the late nonstructural 100,000-dalton protein with newly synthesized hexon in adenovirus type 2-infected cells

Analysis of cellular extracts of HeLa cells infected with adenovirus type 2 (Ad2) by immunoprecipitation with antiserum against the late nonstructural 100,000-dalton (100K) protein revealed the presence of a specific complex between the 100K protein and newly synthesized hexon molecules. Serological analysis of the hexon molecule in the 100K/hexon complex with antibodies specific for hexon monomers or trimers showed that only monomeric hexon molecules were associated with the 100K protein. By immunofluorescence microscopy this monomeric hexon was primarily found in the cytoplasm, whereas the trimeric form was mainly confined to the nucleus of infected cells. We conclude that in the cytoplasm of Ad2-infected cells newly synthesized, monomeric hexon molecules can interact with the 100K protein. This suggests that the 100K protein may play some role either in trimerization of newly synthesized, monomeric hexon molecules and/or in its transport from the cytoplasm into the nucleus.     

Identification of DNA polymerase(s) involved in the repair of viral and cellular DNA in herpes simplex virus type 2-infected cells

When human embryonic fibroblasts (HEF) were infected with herpes simplex virus type 2 (HSV-2), replicative viral DNA synthesis and some repair synthesis of cellular DNA were induced at the early stage of infection, but almost all DNA synthesis at the late stage of infection was derived from repair synthesis of cellular and viral DNA (Y. Nishiyama and F. Rapp, Virology 110, 466-475, 1981). In this study, we have assessed the effects of DNA polymerase inhibitors on repair DNA synthesis HSV-2-infected HEF. Both viral and cellular DNA syntheses during the late stage of infection were extremely resistant to aphidicolin and phosphonoacetic acid but partially sensitive to high concentrations of 1-beta-D-arabinofuranosylcytosine, while replicative viral DNA synthesis during the early stage of infection was very sensitive to all of those inhibitors. The results suggest that neither HSV-induced DNA polymerase nor cellular DNA polymerase alpha was involved in the repair synthesis of viral and cellular DNA but that cellular DNA polymerase beta was.     

Radiation-induced pulmonary fibrosis resolves spontaneously if dense scars are not formed

The relation of static compliance of excised lungs to collagen accumulation and histologic fibrosis was examined in Syrian hamsters inhaling sufficient 238PuO2 particles to achieve initial lung burdens of 50 or 100 nCi. Control animals were exposed to nonradioactive aerosols. Irradiated lungs from hamsters at both dose levels had compliance reduced to the same extent at point of maximal reduction. However, collagen accumulation was more closely related to 238Pu exposure level than the compliance measurements. Histologic examination revealed both diffuse alveolar thickening and some dense fibrous scars, the former predominating at lower dose levels. Hamsters exposed to 50 nCi 238PuO2 showed normal collagen content and static lung compliance with minimal histologic fibrosis 288 days after exposure. In contrast, hamsters exposed to 100 nCi had significant pulmonary fibrosis at that time and the highest incidence of dense scars at any time period. Such findings are consistent with a stiffening of lung parenchyma. They suggest that the diffuse interstitial fibrosis developed by this injury resolves spontaneously; dense fibrous scars, however, do not.     

Identification and partial characterization of a new (50 S) subviral particle in Mengo virus-infected L cells.

A previously undetected subviral particle has been found in Mengo virus-infected L cells by sucrose density gradient centrifugal analysis of cytoplasmic supernatants (S₂₀) prepared from cells after labeling with [³H]amino acids during the early to mid-log phase of virus production. The particle (designated the “50 S particle” from its position between the ribosomal subunits in the gradient), together with mature virions (150 S) and previously described 14 S particles (McGregor et al., 1975), can be recovered from the S₂₀ fraction by high-speed centrifugation. It contains no RNA and is composed of equimolar amounts of the polypeptides ?, α, and γ. The results of conventional pulse-chase experiments suggest that it may be a precursor in the assembly of Mengo virions, but more convincing evidence that this is the case was obtained from experiments in which the chase was carried out in the presence of cordycepin (3′-deoxyadenosine). In the presence of this inhibitor of viral RNA synthesis, there is a significant accumulation of 50 S particles, and when the inhibition in reversed, a quantitative transfer of radiolabel from 50 S particles to mature virions ensues. The recovery of 50 S particles (and of mature virions) from cell homogenates is strongly dependent upon the concentration of KCl in the suspending buffer; only trace amounts are recovered at concentrations of less than 60 mM, while maximum recovery is achieved at a concentration of 100 mM.     

Host range restriction of vesicular stomatitis virus on duck embryo cells.

Vesicular stomatitis virus (VSV) plaque formation and replication are restricted in duck embryo cells compared to chick embryo cells. Mutants of VSV which replicate normally on both chick and duck cells were isolated. The duck-adapted mutant, when passed through chick cells, retains its ability to grow normally on duck cells. This indicates that the ability of this virus to grow on duck cells is due to mutation rather than to host-controlled modification. VSV is restricted in duck cells but not in quail or pheasant embryo cells which follows the evolutionary relationship of these species. Newcastle disease virus and Rous sarcoma virus are restricted in duck cells also. Virus-specific protein synthesis of the wild-type VSV is greatly reduced in duck cells compared to chick cells. A structural protein of the duck cell-adapted mutant is altered.     

Adenovirus-associated virus polypeptides synthesized in cells coinfected with either adenovirus or herpesvirus.

One major and two minor structural polypeptides of adenovirus-associated virus (AAV) were synthesized in AAV-infected cells coinfected with either adenovirus or herpesvirus as helper. The molar proportions of these polypeptides appeared to be the same as those found in the virion. Transport of these polypeptides from the cytoplasm to the nucleus occurred rapidly. No evidence was found for the synthesis of a large precursor protein in either system. Since complete AAV virions are not found in AAV-infected cells coinfected with herpesvirus, these findings indicate a failure in virus assembly in this system. This failure may be expressed at the level of DNA strand segregation or encapsidation of DNA, or in faulty capsid assembly.     

Purification and characterization of deoxythymidine kinase (dTK) induced in dTK- 3T3 mouse cells by equine herpesvirus type 1 (EHV-1).

Infection of mouse 3T3 cells deficient in cytosol deoxythymidine kinase (dTK^?) with the Kentucky A strain of EHV-1 resulted in a 20- to 30-fold increase in cytosol dTK activity. The EHV-1-induced dTK was partially purified from such cells by affinity chromatography on deoxythymidine (dT)-Sepharose and characterized by electrophoretic, enzymatic, and immunological criteria. The purified EHV-1 dTK migrated in polyacrylamide gels with an R_f of 0.25 and sedimented in glycerol gradients with an S value of 5.2, corresponding to a molecular weight of 85,000. Deoxycytidine could not serve as an alternative substrate for the viral-induced enzyme. A rabbit antiserum prepared against EHV-1-infected horse cells neutralized the viral-induced dTK activity purified from infected mouse (dTK^? 3T3) cells, but not the kinase activities from uninfected 3T3 cells. EHV-1 dTK differed from 3T3 cytosol dTK also in the greater resistance of the viral enzyme to feedback inhibition by dTTP and dCTP, its inhibition by arabinosylthymine, and its ability to utilize a variety of nucleoside triphosphates as phosphate donors. The purified EHV-1 kinase could be distinguished from 3T3 mitochondrial dTK by electrophoretic mobility and inhibition by anti-(EHV-1) serum. These results lend further support to the hypothesis that the dTK induced by EHV-1 is coded by the virus genome.     

Monoclonal antibodies to respiratory syncytial virus: Detection of virus neutralization and other antigen-antibody systems using infected human and murine cells

Monoclonal antibodies to human respiratory syncytial (RS) virus-specific antigens can be obtained without preliminary recourse to large-scale culture and purification of the virion. Lytically infected human and persistently infected murine cultured cells expressing RS virus-specific cell surface and cytoplasmic antigens were substituted as priming immunogens and as substrates in solid-phase antibody radioimmunoassays. Seven hybridoma clones secreting murine IgG of either the γ₁ or the γ_2A subclass bearing κ light chains were isolated. Two of the antibodies were specific for cell surface viral antigens, but only one was able to neutralize RS virus infectivity. The five remaining antibodies did not neutralize virus infectivity and were specific for viral antigens associated with large cytoplasmic inclusions as judged by indirect immunofluorescence (IF) analysis on fixed infected cells. Similar IF analysis using live cells revealed that those antigens, associated with the cytoplasmic inclusions in both the human and murine infected cells, were not expressed on the cell surface of the live infected human cells, but were expressed on the cell surface of the live infected murine cells. Monoclonal antibodies generated via the present system will prove useful in the immunological analysis of viral components which are associated with specific pathogenic functions, such as infectivity, and those which may be abnormally exposed at the surface of persistently infected cells.     

Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus-infected tobacco

A ribonucleoprotein fraction (vRNP) of a characteristic buoyant density greater than the buoyant density of tobacco mosaic virus (TMV) particles has been isolated from infected tissue by Cs2SO4 density gradient centrifugation. The vRNP particles appear to be TMV specific because they are synthesized in the presence of actinomycin D and have RNAs identified as genomic and I-class subgenomic (apparent Mr 1.1-1.3 x 106 and 0.60.8 x 10(6)) RNAs by their electrophoretic mobility and hybridization to plasmid-bearing RNA sequences. Polypeptides of apparent molecular weights 17,500 (TMV coat), 31,000, 37,000, and 39,000 were major constituents of vRNP. Of the minor polypeptides, those of apparent molecular weights 70,000, 68,000, 55,000, and 25,000 had electrophoretic mobilities similar to mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. vRNP from common TMV-infected plants, but not from plants infected with a mutant that did not form native coat protein, reacted with immunoglobins against TMV and TMV coat protein. Common TMV and its vRNP differed in the extent of reactivity toward the two immunoglobins, in electron microscopic appearance, and in the higher sensitivity of vRNP to ribonuclease.     

Replication of herpesvirus DNA. VI. Virions containing either isomer of pseudorabies virus DNA are infectious

Pseudorabies virus DNA exists in two isomeric forms in which the short unique sequence is present in two orientations with respect to the long unique sequence. The viral DNA present in the virions of 21 individual plaques was analyzed. In all cases, equimolar amounts of the two isomeric forms of the DNA were present, indicating that isomerization of the DNA is a rapid process which is complete by the time a small plaque (<2 mm) has developed. Virions containing either isomeric form of the DNA adsorb equally well to cells and either isomeric form of the DNA has the same likelihood of becoming associated with the cell nucleus and to form circles (or concatemers) before initiation of DNA synthesis. The two isomeric forms of viral DNA are also equally represented in the genomes that mature first from concatemeric replicating DNA in cells infected at low multiplicities (0.01 PFU/cell). Furthermore, during the first round of DNA replication in cells infected at low multiplicity, equimolar amounts of the two isomeric forms of the DNA replicate. Since, in this experiment, each cell was infected with a maximum of one viral particle per cell, we conclude that virions containing either isomeric form of the DNA can initiate infection. Previous data (J. M. DeMarchi, T. Ben-Porat, and A. S. Kaplan, 1979, Virology97,457–463) have indicated that each cell, in which infection is initiated, is able to produce a plaque. We therefore conclude that virions containing either isomeric form of the DNA are infectious.     

Inhibitor of virus replication released from tobacco mosaic virus-infected protoplasts of a local lesion-responding tobacco cultivar

A substance(s) inhibiting virus replication (IVR) is released into the medium from tobacco mosaic virus-infected protoplasts of a cultivar in which the infection in the intact plant is localized. IVR inhibited virus replication in protoplasts from both local lesion-responding resistant, Samsun NN and systemic-responding susceptible, Samsun plants, when applied up to 18 hr after inoculation. It was not produced in protoplasts from susceptible plants nor from noninoculated protoplasts of the resistant cultivar. IVR was partially purified using ZnAc2 precipitation, and yielded two biologically active principles with molecular weights of about 26,000 and 57,000, as determined by gel filtration.