Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.     

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacteriol. 92:1792-1804. 1966.-After the development of marked cytopathic effects in HeLa cultures infected with the Edmonston strain of measles virus, renewed cell growth occurred, and HeLa sublines persistently infected with measles virus were obtained. Persistent infection has occurred in a large fraction of the cells of infected clonal lines for more than 300 to 500 cell generations during a period of 6 years. One mechanism by means of which infection was maintained in the clonal lines is transmission of virus or viral subunits from cell to cell at division. Continued subculture of the persistently infected populations resulted in the virtual disappearance of cytopathic effects, a marked decrease in the amount of extracellular virus, and alterations in the cytopathogenicity of virus recovered from persistently infected populations. The intracellular virus-host cell events in late passages of the infected clonal lines appeared to be similar to those in cells of primary infected cultures at early stages of infection, as judged by the pattern of viral immunofluorescence and the very low incidence of cells with intranuclear inclusion bodies. Cultures of the persistently infected clonal lines were highly resistant to super infection by parent Edmonston virus. Cultures of one of these clonal lines were just as susceptible as normal HeLa cultures to vaccinia, herpes simplex, and polio type 2 viruses, and a simian agent, with a possible low degree of resistance to the simian agent.     

Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.     

Leader sequence distinguishes between translatable and encapsidated measles virus RNAs.

The 3'-terminal 55 nucleotides of the negative-strand measles virus RNA genome called the leader sequence is not transcribed into a detectable distinct RNA product. Most of the monocistronic N and bicistronic N-P RNAs lack the leader sequence. However, a subpopulation of the N and N-P RNAs and all of the antigenomes possess this leader. Here, we show that leader-containing subgenomic RNAs are functionally distinct from their leaderless counterparts. In measles virus-infected cells, leaderless monocistronic N and bicistronic N-P RNAs were associated with polysomes. By contrast, leader-containing N and N-P RNAs were found exclusively in nonpolysomal ribonucleoprotein complexes that were resistant to RNase and had a buoyant density of 1.30 g/ml, the same as that of antigenomic ribonucleoprotein complexes. Both antigenomic and subgenomic ribonucleoprotein complexes were specifically immunoprecipitated by antiserum against the N protein, and leaderless RNAs were not found in these complexes. These findings suggest that measles virus distinguishes RNAs destined for encapsidation or translation by the presence or absence of a leader sequence.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

Measles Virus-Specified Polypeptide Synthesis in Two Persistently Infected HeLa Cell Lines

Measles virus-directed protein synthesis was examined in two HeLa cell lines (K11 and K11A) that are persistently infected with wild-type measles virus. Four viral proteins (H, hemagglutination protein; P, nucleocapsid-associated protein; NP, the major nucleocapsid protein; and M, the matrix protein) were readily detected in both cell lines by immune precipitation of [(35)S]methionine-labeled cell extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three (H, NP, and M) of the four viral proteins in both K11 and K11A cells differed from the corresponding viral proteins synthesized in HeLa cells acutely infected with the parental wild-type virus. In addition, the M protein from K11A cells migrated significantly more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the M protein from K11 cells, and there appeared to be slight differences in the H and NP proteins between these two persistently infected cell lines. The altered viral proteins detected in K11 and K11A cells appeared to be the result of viral mutations rather than changes in the host cell, since virus recovered from these cells directed the synthesis of similar aberrant viral proteins in HeLa cells. Virus recovered from K11 cells and virus recovered from K11A cells were both temperature sensitive and grew more slowly than wild-type virus. HeLa cells infected with virus recovered from K11 cells readily became persistently infected, resembling the original persistently infected K11 cells. Thus, viral mutations are associated with persistent measles virus infections in cell cultures.     

Subgenomic HCV RNA replication and its localization in the nucleus of the infected cells

Cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. It is known that HCV contains proteins which interact with host cell proteins.To see whether a HCV RNA replicon can interact in the same way with host cell proteins, HCV RNA replicon was transfected in Huh7 cells. In most infected cells, HCV replicon is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own is positive for NS3. The presence of NS3 in the nuclei of Huh7 cells indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.Keyword: Hepatitis C Virus