The intracellular growth of bacteriophages. I. Liberation of intracellular bacteriophage T4 by premature lysis with another phage or with cyanide

A method is described for liberating and estimating intracellular bacteriophage at any stage during the latent period by arresting phage growth and inducing premature lysis of the infected cells. This is brought about by placing the infected bacteria into the growth medium supplemented with 0.01 M cyanide and with a high titer T6 lysate. It was found in some of the later experiments that the T6 lysate is essential only during the first half of the latent period. Cyanide alone will induce lysis during the latter part of the latent period. Using this method on T4-infected bacteria it is found that during the first half of the latent period no phage particles, not even those originally infecting the bacteria, are recovered. This result is in agreement with the gradually emerging concept that a profound alteration of the infecting phage particle takes place before reproduction ensues. During the second half of the latent period mature phage is found to accumulate within the bacteria at a rate which is parallel to the approximately linear increase of intracellular DNA in this system. However, the phage production lags several minutes behind DNA production. When 5-methyltryptophan replaced cyanide as the metabolic inhibitor, similar results were obtained. The curves were, however, displaced several minutes to the left on the time axis. The results are compared with Latarjet's (16) data on x-radiation of infected bacteria and with Foster's data (18) concerning the effect of proflavine on infected bacteria. Essential agreement with both is apparent.     

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.     

Intracellular growth of bacteriophage studied by roentgen irradiation

Growing Escherichia coli infected with bacteriophage T2 was x-rayed during the 21 minute latent period which elapses between infection and lysis of the cells. Survival curves of the infected bacteria were determined almost from minute to minute; they disclosed the following facts which are related to the process of phage growth: During the first 7 minutes, the infective virus particle remains in the cell unique and genetically intact. The host cell synthesizes some ultraviolet-absorbing material probably devoted to building future particles. From the 7th to 9th minute the x-ray resistance of the virus particle increases, probably because of some internal change. Then, multiplication starts and is completed at about the 13th minute, when an average of 130 virulent units is present per cell, displaying an x-ray resistance twice as high as that of the extracellular virus particle. From 13 minutes to the end, the new units progressively recover the x-ray sensitivity of the extracellular virus. Nothing can be said about either the rate of multiplication between 9 and 13 minutes, or the nature of the multiplying units, except that they are more radiation-resistant (probably smaller) than the extracellular virus. The first steps of the growth process are favored by an unknown component of the lysate, different from the active particles. Several particles can grow in the same host cell.     

Lysis Inhibition in Escherichia coli Infected with Bacteriophage T4

A technique of continuous filtration of T4-infected Escherichia coli has been devised to study the phenomenon of lysis inhibition. Studies using this technique revealed that the length of the lysis delay caused by superinfection can attain only certain discrete values, which for low average multiplicity of superinfection is thought to be a reflection of the actual number of superinfecting particles per cell. The time interval between primary and superinfection had little effect on the length of lysis delay. With increasing rate of superinfection, the length of lysis delay decreased. In superinfected cells, the concentration of endolysin exceeded the final concentration in nonsuperinfected cells. Superinfection of a lysing culture induced lysis inhibition immediately. Temperature-shift experiments, with cells primarily infected by a temperature-sensitive endolysin mutant, revealed that after the normal latent period superinfection was unable to induce lysis inhibition. Amber-restrictive cells, which were primarily infected by an endolysin negative amber mutant, released adenosine triphosphate (ATP) at the end of the normal latent period although lysis did not occur. Superinfection reduced the loss of ATP markedly. The hypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.     

Inhibition of lactic streptococcus bacteriophage by crystal violet and other agents

Ninety-nine selected compounds and eleven antibiotic-producing organisms were tested for antiphage activity and host toxicity. A paper disc-agar diffusion method was used for primary screening and quantitative methods were employed for confirmatory investigation. Most of the agents tested, although previously reported as inhibitory to one or more other virus-host systems, did not selectively prevent multiplication of lactic streptococcus bacteriophage. Several compounds which prevented mass lysis were extremely toxic to host bacteria. Crystal violet suppressed growth of two phage strains at a level (1.0 x 10(-7)M) which permitted normal growth of the host cells. Failure of crystal violet to prevent multiplication of many phage strains suggested possible variations in the multiplication mechanisms of different strains of virus. Virustatic levels of crystal violet did not destroy unadsorbed virus, reduce adsorption, or prevent invasion; increase of virus was reduced in one-step growth experiments; mass lysis was prevented or delayed in long time experiments. Addition and removal of crystal violet at various intervals during the latent period resulted in virus yields directly related to the portion of the latent period during which no dye was present. Duration of the latent period was unaffected. Single burst experiments indicated that the yield of plaque-forming particles per infected bacterium was reduced; the proportion of infected bacteria giving rise to active progeny did not appear to be influenced to a significant degree. Crystal violet apparently interferes with intracellular multiplication of the virus, possibly by combination of the dye with phage DNA or fractions thereof at some critical stage in the incorporation of DNA into the virus particle.     

Biological consequences of infection of Escherichia coli B by alkylated T7 bacteriophage.

Alkylation of T7 bacteriophage considerably delayed phage development and reduced the phage's killing action on host cells. Only a small fraction of infected cells produced phage. For these phages, the latent period was markedly prolonged but the burst was equivalent to or only slightly lower than that of untreated phage. In the progeny of alkylated phage, there was an increase in the fraction of defective particles as well as a change in their morphology. These data show that infection with alkylated T7 bacteriophage is to a large degree abortive; hence, biological consequences of this infection are very different from those characteristic of a normal virus infection.     

Influence of urethane and of hydrostatic pressure on the growth of bacteriophages T2, T5, T6, and T7

In 0.5 per cent NaCl, nutrient broth at 35 degrees C., urethane in a concentration of 0.4 M stops the reproduction of Escherichia coli, strain B. On dilution with 20 volumes of sterile medium, growth is resumed at its former rate after a short lag. In the one-step growth of T2, 15, T6, or T7, in the same medium at the same temperature, 0.4 M urethane, when added at the time of infection, had no apparent effect on adsorption and caused no decrease in titer throughout the latent period of the control, but completely prevented a rise in titer. If diluted 1:20 with sterile medium prior to a certain critical time in the latent period, however, bacteriophage was liberated at the same time, and in the same amount as in the control. The initial stage of apparent insensitivity to the drug lasts from the time of infection until the approximate critical times of 7 minutes with T7, T2, or T6, or 13 minutes with T5. Under the conditions described, the normal latent periods were 14, 23, 30, and 44 minutes for T7, T2, T6, and T5, respectively. At the critical times referred to above, there begins a stage characterized by complete sensitivity, rather than complete insensitivity, to 0.4 M urethane, in the sense that no active phage is subsequently liberated in continued presence of the drug. The length of this completely sensitive stage, as judged by addition of the drug at successive intervals during the latent period, extends from approximately 7 until 9 minutes after infection with T7, 7 until 15 minutes with T2 or T6, or 13 until 25 minutes with T5. When the urethane is added late in this stage of T2, a decrease in initial titer takes place as judged by assays made 40 minutes after infection, the maximum effect occurring when the drug is added between 14 and 15 minutes after infection. When added subsequently to the completely sensitive stage of each type, i.e. subsequently to 9 minutes after infection with T7, 15 minutes with T2 or T6, or 25 minutes with T5, liberation of the bacteriophage takes place in presence of the drug, but the yield is reduced, the amount of reduction being greater the sooner it is added. The yield increases as addition of the drug is delayed, but it is measurably reduced when added late in the rise period. Macroscopic lysis with T7 is delayed by 0.4 M urethane, when present from the time of infection. The delay is less with increased multiplicities of infection. A similar delay occurs with T6r at a multiplicity of 4. The application of hydrostatic pressures of 7,000 to 9,000 p.s.i. early in the latent period, within 5 to 8 minutes after infection, prevents a yield in each of the four phage types, and if maintained for lengthy periods of time a reduction in initial titer occurs. If released at various times shortly after the latent period, a rise in the titer occurred after a certain interval whose length was characteristic of the phage type. The yield was less the longer the release of pressure was delayed. When the pressure was first applied late in the latent period, large amounts of phage were liberated either under pressure or explosively when pressure was released to make the assays. Hydrostatic pressure at 6,000 p.s.i. had little effect on the rate or amount of macroscopic clearing with T7 in relatively high multiplicity of infection, when applied at the start of lysis, but slowed the rate and reduced the amount of clearing when applied shortly after infection.     

Isolation and Characterization of a Bacteriophage Tail-Like Bacteriocin from a Strain of Rhizobium

Bacterial strain 16-3 spontaneously produces a bacteriocin which inhibits the growth of closely related strain 16-2. Both strains were newly isolated from root nodules of lupines and probably belong to the species Rhizobium lupini. Production of infectious progeny of newly isolated virulent phage 16-2-4 in strain 16-2 is inhibited completely if complexes are bacteriocin-treated during the first half of the latent period. Treatment begun during the second half leads to premature lysis of complexes and inactivates only those progeny phages which were not yet fully matured at the moment of the particle-induced lysis. Examination by electron microscope of the bacteriocin enrichment revealed the presence of particles 123 nm in length which resemble the tails of T-even bacteriophages. Since the particles sediment together with the bactericidal activity in the sucrose gradient and adsorb specifically to bacteriocin-sensitive cells, it is concluded that they are identical with the bactericidal agent. The particles are not found attached to phage heads and cannot self-propagate; they are regarded as incomplete and are named INCO particles. INCO particles consist of a core enveloped by a contractile sheath. One end of the sheath is connected to a baseplate to which six fibers, each 32 nm in length, are attached. These connect the baseplate of an adsorbing particle to the cell surface. Since INCo cores are probably empty, it is concluded that specific adsorption of the particles to the bacterial surface is sufficient to inactive sensitive cells irreversibly.     

The effect of iodoacetate and other inhibitors on phage production and lysis in three phage systems

The minimal bacteriostatic concentration of iodoacetate, azide, or proflavine was added at intervals during the latent periods of virus in three different bacterium-bacteriophage systems (M. aureus, B. mycoides, E. coli). For each interval at which inhibitor was added, the occurrence of lysis and the final yield of phage were determined. In the B. mycoides and E. coli systems, when added during the first part of the latent period, inhibitor prevented lysis and no phage was released. Introduction of inhibitor during the last part of the latent period resulted in normal lysis and in a linear increase in phage that progressively approached the yield obtained in the absence of inhibitor (the later the introduction, the higher the yield). In the M. aureus system, phage production and lysis in the presence of inhibitor followed the same general pattern, except that release of phage and normal lysis occurred in infected cells to which inhibitor had been added quite early in the latent period. Our results, when compared with those of Foster (1948) with proflavine and Bozeman et al. (1954) with chloramphenicol, suggest that (1) the final phage yields represent the amount of mature intracellular virus present at the time of addition of inhibitor and (2) the reactions leading to lysis proceed independently of those leading to the formation of mature virus once phage infection has reached a critical point in time.     

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.     

Cell cycle dependency of murine cytomegalovirus replication in synchronized 3T3 cells.

Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host S-phase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidine-induced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle.     

The effect of various culture media on infection,growth, lysis,and phage production of B. megatherium

B. megatherium cells were grown in various culture media, centrifuged and washed, and suspended in other culture media containing "C" or "T" phage. The per cent of infection, rate of growth, lysis, and phage production were determined. The behavior of the system depends on the culture medium in which the cells were grown and also on the culture medium in which they were mixed with phage. With the T phage it is possible to set up systems which yield the following results: 1. No infection, normal growth, no phage production. 2. Infection, normal growth, no lysis) phage production. 3. Infection, growth for several hours, lysis, and phage production. 4. Infection, no growth, lysis, and phage production. The C phage system is less affected by changes in the culture medium. The change in the behavior of the cells with T phage probably is not due to selection since it occurs without much growth of the culture, and is readily reversible.     

The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

HIV fusion and entry into CD4 T cells are mediated by two receptors, CD4 and CXCR4. This receptor requirement can be abrogated by pseudotyping the virion with the vesicular stomatitis virus glycoprotein (VSV-G) that mediates viral entry through endocytosis. The VSV-G-pseudotyped HIV is highly infectious for transformed cells, although the virus circumvents the viral receptors and the actin cortex. In HIV infection, gp120 binding to the receptors also transduces signals. Recently, we demonstrated a unique requirement for CXCR4 signaling in HIV latent infection of blood resting CD4 T cells. Thus, we performed parallel studies in which the VSV-G-pseudotyped HIV was used to infect both transformed and resting T cells in the absence of coreceptor signaling. Our results indicate that in transformed T cells, the VSV-G-pseudotyping results in lower viral DNA synthesis but a higher rate of nuclear migration. However, in resting CD4 T cells, only the HIV envelope-mediated entry, but not the VSV-G-mediated endocytosis, can lead to viral DNA synthesis and nuclear migration. The viral particles entering through the endocytotic pathway were destroyed within 1–2 days. These results indicate that the VSV-G-mediated endocytotic pathway, although active in transformed cells, is defective and is not a pathway that can establish HIV latent infection of primary resting T cells. Our results highlight the importance of the genuine HIV envelope and its signaling capacity in the latent infection of blood resting T cells. These results also call for caution on the endocytotic entry model of HIV-1, and on data interpretation where the VSV-G-pseudotyped HIV was used for identifying HIV restriction factors in resting T cells.     

Amber Mutants of Bacteriophages T3 and T7 Defective in Phage-directed Deoxyribonucleic Acid Synthesis

Amber mutants of the related phages T3 and T7 were isolated and tested for their ability to restore-as the wild type does-thymidine incorporation in ultraviolet (UV)-irradiated, UV-sensitive, nonpermissive host bacteria (Escherichia coli B(s-1)). Most amber mutants had this ability. However, in both T3 and T7, mutants unable to promote thymidine incorporation under these conditions were found and classified into two well-defined complementation groups: T3DO-A and T3DO-B, T7DO-A and T7DO-B. Infection of B(s-1) cells with representatives of groups DO-A had the following characteristics: (i) phage-directed uridine uptake in UV-irradiated cells was reduced to less than 20% of normal; (ii) breakdown of host deoxyribonucleic acid (DNA) was delayed and incomplete; (iii) no serum-blocking antigens appeared; (iv) no cell lysis occurred; (v) the ability to exclude the heterologous wild type was impaired. Amber mutants of the DO-B groups, infecting B(s-1), were able to: (i) promote an efficient phage-directed uridine uptake in UV-irradiated cells; (ii) bring about rapid breakdown of host DNA; (iii) synthesize serum-blocking antigens; (iv) lyse the host cells, generally after the normal latent period; (v) exclude efficiently the heterologous wild type. Although physiological similarities between the respective DO-A mutants or DO-B mutants of T3 and T7 were evident, no physiological cross-complementation occurred, and genetic crosses gave no evidence of genetic homologies between groups of T3 and T7.     

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Exclusion of T4 phage by the hok/sok killer locus from plasmid R1.

The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and lambda phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by approximately 85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior.     

Phage formation in Staphylococcus muscae cultures. X. The relationship between virus synthesis,the release of bacterial ribonucleic acid,virus liberation,and cellular lysis

1. Under a variety of conditions in which cells are infected with one or a few virus particles and the host cells are killed, but no infective particles or virus material is formed as indicated by plaque count, one-step growth curve, or protein or desoxyribonucleic determinations, the cells neither lyse nor release ribonucleic acid into the medium. 2. The "killing" effect of S. muscae phage is separate from its lytic property. 3. The release of ribonucleic acid into the medium is not simply due to the killing of the cell by the virus, and ribonucleic acid is never found in the medium unless virus material is synthesized. 4. Infected cells of S. muscae synthesizing virus release ribonucleic acid into the medium before cellular lysis begins and before any virus is liberated. 5. The higher the phage yield the more ribonucleic acid is released into the medium before any virus is released. 6. Phage may be released from one strain of Staphylococcus muscae without cellular lysis, although bacterial lysis begins shortly after the virus is released. In another strain, infected under similar conditions, virus liberation occurs simultaneously with cellular lysis. 7. The viruses liberated from both bacterial strains appear to be the same in so far as they cannot be distinguished by serological tests, have the same plaque type and plaque size, and need the same amino acids added to the medium in order to grow. Furthermore, the virus liberated from one strain can infect and multiply in the other strain and vice versa. 8. It is suggested that virus synthesis, in S. muscae cells infected with one or a few phage particles, leads to a disturbance of the normal cellular metabolism, resulting in lysis of the host cell.     

The amino-terminal functions of the simian virus 40 large T antigen are required to overcome wild-type p53-mediated growth arrest of cells.

High levels of the p53 tumor suppressor protein can block progression through the cell cycle. A model system for the study of the mechanism of action of wild-type p53 is a cell line (T64-7B) derived from rat embryo fibroblasts transformed by activated ras and a temperature-sensitive murine p53 gene. At 37 to 39 degrees C, the murine p53 protein is in a mutant conformation and the cells actively divide, whereas at 32 degrees C, the protein has a wild-type conformation and the cells arrest in the G1 phase of the cell cycle. Wild-type simian virus 40 large T antigen and a variety of T-antigen mutants were assayed for the ability to bypass the cell cycle block effected by the wild-type p53 protein to induce colony formation at 32 degrees C. The results indicate that two functions within the amino terminus of T antigen are essential to induce cell growth: (i) the ability to bind to the retinoblastoma protein, Rb, and (ii) the presence of a domain in the first exon that appears to interact with the cellular protein, p300. Thus, the cell cycle arrest triggered by wild-type p53 may be overcome by formation of a T-antigen complex with Rb, p300, or both that could then function to either remove p53-mediated negative growth regulatory signals or promote a positive cell growth signal. Surprisingly, T antigen-p53 complexes are not required to overcome the temperature-sensitive p53 block to the cell cycle in these cells. These data suggest that simian virus 40 T antigen associated with Rb, p300, or both proteins can communicate in a cell with the functions of the wild-type p53 protein.     

LPP-1 Infection of the Blue-Green Alga Plectonema boryanum: I. Electron Microscopy

One-step growth and intracellular growth experiments were performed at high multiplicities of the virus LPP-1 during the infection of the blue-green alga Plectonema boryanum. The eclipse period lasts until 4 hr after infection, the latent period terminates at 6 hr, and the rise period continues until 14 to 16 hr after infection. The burst size was independent of multiplicity of infection over the ranges from 1 to 50. The burst size was 3,000 to 5,000 plaque-forming units (PFU) per infectious center or about 200 PFU per cell. Samples for electron microscopy were taken at characteristic times during the lytic cycle. The first sign of viral infection was the invagination of the photosynthetic lamellae at 3 hr after infection. Mature virions were visible at 4 hr. By 6 to 7 hr, many mature intracellular viral particles could be seen, with lysis beginning at 7 hr. By 10 hr after infection, all infected cells contained mature virions. No evidence for mass migration of performed viral precursors was obtained. The invagination of the lamellae could be prevented by the early addition of chloramphenicol, which implies that this process requires protein synthesis.