Yaba tumor poxvirus synthesis in vitro. 3. Growth kinetics

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Victoria A. Haendiges, and Etienne de Harven. Yaba tumor poxvirus synthesis in vitro. III. Growth kinetics. J. Bacteriol. 91:1986-1991. 1966.-The infectious synthetic cycle of Yaba poxvirus by BSC-1 cells at 35 C is described and discussed together with related observations obtained by immunofluorescence and electron microscopy. After adsorption of virus at 25 C, at least 6 hr of incubation at 35 C was required before viral eclipse was demonstrable. Infectious progeny appeared after an additional 48 hr of incubation at 35 C. Newly synthesized virus particles were seen at 68 hr but not at 44 hr by electron microscopy. Maximal maturation occurred between 100 and 120 hr. Maximal yields were obtained from the 5th through the 8th day. The differences in kinetics between Yaba and vaccinia poxviruses, as described for the latter in the literature, are discussed. Possible factors accounting for these differences are enumerated.     

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.     

Growth Characteristics of Kilham Rat Virus and Its Effect on Cellular Macromolecular Synthesis

Kilham rat virus (KRV) is adsorbed into the rat nephroma cell within 1 hr after infection. There follows a latent period of about 12 hr during which less than 1% of the input infectious virus can be accounted for. New infectious virions can be detected at about 12 hr and the maximal yield of virus is attained by 23 hr after infection. The increase in final virus yield is about 200-fold over that found in the latent period. During this 23-hr period of virus growth, the rate of protein synthesis remains 75 to 100% of that in the uninfected cell. Ribonucleic acid (RNA) synthesis during this period is maintained at 100 to 150% of that found in the control cells. The addition of the inhibitor of deoxyribonucleic acid (DNA) synthesis, 5-fluoro-deoxyuridine (FUDR), up to 8 hr after infection completely suppresses virus production. After 8 hr, viral DNA production has started and FUDR inhibition progressively decreases until by 23 hr the addition of the inhibitor no longer causes a reduced virus yield. Viral DNA synthesis once initiated is required for the remainder of the 23-hr virus cycle. Viral DNA synthesis probably begins about 4 hr before the production of infectious virions. In the KRV-infected cells, DNA synthesis decreased sharply for 6 to 7 hr after infection in comparison to the uninfected cell. At 7 to 8 hr after infection, DNA synthesis in the infected cell increased and was maintained at a higher level than in the control cells for the rest of the virus growth period.     

Virological, Immunochemical, and Cytochemical Studies of Four HeLa Cell Lines Infected with Two Strains of Influenza Virus

The production of infectious virus, hemagglutinin, and viral (V) antigens and the changes in ribonucleoprotein (RNP) and lipoprotein metabolism have been studied in four sublines of HeLa cells infected with the PR8 and a PR8 recombinant strain of influenza virus. Much greater amounts of infectious virus and much less hemagglutinin were produced by the PR8 recombinant than by PR8 virus in all four cell lines. Different amounts of infectious virus per infected cell were produced by the recombinant in the four cell lines, whereas very little infectious virus was produced by the PR8 strain in any of the HeLa cells. In all cell lines infected with both strains of virus, "soluble" (S) antigen appeared early in the nucleolus. In cells infected with PR8 recombinant, S antigen subsequently filled the nucleus and later appeared in the cytoplasm. In most cells infected with PR8 virus, nuclear S antigen did not fuse to fill the nucleus, and S antigen was not detected in the cytoplasm. V antigen was observed in the cytoplasm of cells when diffuse nuclear S antigen had formed. The earliest and most frequent change in the RNP of the infected cells was a decrease in stainable RNP spherules (nucleolini) in the nucleolus. This was followed, in a smaller proportion of cells, by the appearance of nuclear and cytoplasmic inclusions containing RNP. There was a characteristic difference in the morphology of the cytoplasmic inclusions produced by the two strains of virus, but the same types of inclusions were observed in all four HeLa lines. A significant increase in lipoprotein was observed only in association with the cytoplasmic inclusions produced by PR8 recombinant virus. There was a striking difference in the proportion of cells with cytochemical changes in RNP in the four cell lines. A significant cytopathic effect (CPE) was observed only in three virus-cell systems in which a high proportion of cells exhibited changes in nucleolinar RNP. It is suggested that disappearance of RNP in the nucleolini may be an indication of shutdown of host ribonucleic acid synthesis and that this in turn results in a CPE. Virus infection resulted in a C-mitotic block that was followed by karyorrhexis. Infection of the cell did not always result in the production of infectious virus, in changes in the RNP of the nucleolini, in the development of nuclear or cytoplasmic RNP inclusions, or in CPE. The results suggest that production of infectious virus, shutdown of cellular RNP synthesis with accompanying CPE, and the formation of inclusions appear to be independent events.     

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.     

Effect of Mengovirus Replication on Choline Metabolism and Membrane Formation in Novikoff Hepatoma Cells

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.     

Nonproductive Infection of Guinea Pig Cells with Human Cytomegalovirus

Human cytomegalovirus was capable of adsorbing to and penetrating guinea pig cells, but was unable to replicate new virus. Cultures infected with virus inoculum of high titer showed a cytopathic effect (CPE) characterized by cell rounding. This CPE depended upon the presence of infectious virus, and its extent was directly related to the multiplicity of infection. Staining by indirect immunofluorescence by using human convalescent sera was positive as early as 4 h postinfection. Maximal fluorescence was observed 24 h postinfection when 50% of the cells contained fluorescent antigens both in nuclei and cytoplasm. No evidence for viral replication was found, and no defective particles were detected by electron microscopy. Treatment with actinomycin D or with cycloheximide strongly inhibited both the fluorescent antigens and the CPE, whereas 5-fluorodeoxyuridine and bromodeoxyuridine were ineffective.     

Reovirus-directed Ribonucleic Acid Synthesis in Infected L Cells

Reovirus replication in L-929 mouse fibroblasts was unaffected by 0.5 mug of actinomycin per ml, a concentration which inhibited cell ribonucleic acid (RNA) synthesis by more than 90%. Under these conditions of selective inhibition, the formation of both single-stranded and double-stranded virus-specific RNA was detected beginning at 6 hr after infection. The purified double-stranded RNA was similar in size and base composition to virus RNA and presumably was incorporated into mature virus. The single-stranded RNA formed ribonuclease-resistant duplexes when annealed with denatured virus RNA but did not self-anneal, thus indicating that it includes copies of only one strand of the duplex. The single-stranded RNA was polyribosome-associated and may function as the virus messenger RNA. Production of both types of virus-induced RNA required protein synthesis 6 to 9 hr after infection. At later times in the infectious cycle, only double-stranded RNA synthesis was dependent on continued protein formation.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.     

The course of giardiavirus infection in the Giardia lamblia trophozoites

The subcellular distribution of Giardia lamblia virus RNA in infected G. lamblia trophozoites was examined by in situ hybridization using biotinylated DNA probe and riboprobe. In G. lamblia Portland I strain, which is chronically infected by G. lamblia viruses, the viral RNA was detected in the cytoplasm as well as in the twin nuclei. When riboprobe was used to examine the course of virus infection in WB strain, accumulation of viral RNA was detected only in the cytoplasm prior to the first 72 hr of infection. Using DNA probe, further accumulation of viral RNA in increasing number of cells occurred after the 72nd hr of infection, with the RNA found in both the cytoplasm and nuclei. Eventually, the cell nuclei showed damaged morphology that deteriorated rapidly toward the final stage of infection. These observations indicate that early phase of viral RNA replication may take place in the cytoplasm of infected G. lamblia, but the nuclei are also involved during the late phase of viral replication.     

Iridescent virus replication: patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6

The patterns of nucleic acid synthesis in insect cells infected with iridescent virus types 2 and 6 has been examined using nucleic acid hybridization techniques. Virus-specific RNA synthesis was detected 24 hr after infection. Virus-specific DNA synthesis was detected 96 hr after infection. Host-specific nucleic acid synthesis declined throughout infection, and host-specific nucleic acid synthesis was detected only in the first 48 hr of infection. The synthesis of iridescent virus progeny DNA molecules precedes the appearance of mature iridescent virus particles.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Intracellular development of membrane protein of influenza virus.

The intracellular development of membrane protein (MP) of influenza A virus was investigated by immunofluorescent staining. Monospecific antiserum was prepared by immunizing rabbits with MP eluted from SDS-polyacrylamide gels of SDS-disrupted NWS virions. In the productive infection in clone 1-5C-4 cells, MP antigen was first detected over the whole cell at 4 hr after infection, concomitantly with the appearance of hemagglutinin (HA) antigen in the cytoplasm, and bright nuclear fluorescence was then observed. Nucleoprotein (NP) antigen was detected in the nucleus prior to the appearance of fluorescence of MP antigen and thereafter the cytoplasmic fluorescence developed. Late in infection, all of these three antigens were observed predominantly in the cytoplasm with stronger fluorescence at the cell surface. Essentially similar findings were obtained in the abortive infections in L cells and BHK cells. The above results suggest that the membrane protein of influenza A virus is present in the nucleus as well as in the cytoplasm of infected cells.     

Ribonucleic Acid and protein synthesis in chick embryo cells infected with fowl plague virus

Fowl plague virus comprised four major protein components and several minor ones, two strains of the virus giving similar results. One of the components was identified as the nucleocapsid protein. Synthesis of the virion proteins could readily be detected in infected cells 3 hr after infection. The two subcellular fractions associated with viral ribonucleic acid (RNA) polymerase activity (nuclei and ribosomal pellet) were associated with the protein of the nucleocapsid and a second virion protein of unidentified function. Measurement of viral RNA and protein synthesis in cells infected with preparations of ultraviolet irradiated virus showed that the capacity to synthesise the RNA and protein species of highest molecular weight was lost most quickly, suggesting that the pieces of viral RNA function independently.     

Replication of IPN virus: a cytochemical and biochemical study in SWT cells.

Although IPN virus failed to multiply at 30 degrees, it replicated at 16 degrees and 22 degrees in SWT cells. At 22 degrees the viral eclipse period lasted nearly 6 hr with maximal virion titers attained by 24 hr, whereas replication at 16 degrees was much slower. The replication of the virion was inhibited by 0.05 mug/ml of AD which did not interfere with the production of reovirus. Biochemical studies revealed that cellular DNA synthesis was markedly reduced (greater than 50%) soon after infection whereas total RNA synthesis was enhanced. The period of rapid increase in RNA synthesis paralleled the exponential production of infectious virus. Viral inclusion bodies, revealed by acridine orange-staining of virus-infected cells (SWT and RGG-2) late in the infectious cycle, were found to contain single-stranded RNA on the basis of their staining characteristics and sensitivity to RNase.     

Latency of Human Measles Virus in Hamster Cells

A latent system employing measles virus (Schwarz strain) was developed in hamster embryo fibroblasts (HEF). Measles virus-specific antigen was detected by immunofluorescence in 30 to 50% of HEF cells, and these cells released infectious virus when co-cultivated with a susceptible monkey cell line, BSC-1 cells. No infectious virus could be detected in the cells when measures were taken to exclude passage of viable latent cells onto the indicator BSC-1 cells. Infectious center assays demonstrated that about 1 in 10 of the latently infected cells in the population could release infectious virus. Infectious virus appeared within 6 hr after co-cultivation of the HEF cells with BSC-1 cells, as compared to 24 hr required for normal replication of measles virus in the BSC-1 cells. Furthermore, labeling of progeny virus ribonucleic acid (RNA) by using tritiated uridine, and inhibition of RNA or protein synthesis by 5-azacytidine or cycloheximide suggested that neither additional RNA nor protein synthesis is required after co-cultivation of the cells to effect early virus release. It can therefore be postulated that there is a block at a late step in virus replication in the latently infected hamster cells. The most obvious site would concern maturation of infectious virions at the cell membrane.