Inhibition of Host Protein Synthesis in Type 5 Adenovirus-infected Cells

The effect of type 5 adenovirus infection on the synthesis of host-cell proteins by suspension cultures of KB cells was investigated. Although total protein synthesis continued at a constant rate for approximately 36 hr, net synthesis of five host enzymes (lactic dehydrogenase, acid phosphatase, deoxyribonuclease, fumarase, and phosphoglucose isomerase) was found to stop 16 to 20 hr after infection. The synthesis of alkaline phosphatase stopped 9 to 12 hr after infection. The inhibition of host protein synthesis occurred shortly after the synthesis of viral antigens had begun, accounting for the continued synthesis of total protein. An investigation of the relationship between synthesis of viral antigens and inhibition of host protein synthesis yielded results which suggest that the two processes are in some way coupled.     

Inhibition of the Synthesis of Simian Virus 40 Antigens in Cells Preinfected with Yaba Tumor Virus

The synthesis of tumor and viral antigens after infection of an established line of cynomolgus monkey kidney cells with simian virus 40 (SV40) was compared in cells previously infected with Yaba virus and in cells not preinfected. SV40 failed to induce synthesis of tumor or viral antigens in cells preinfected with Yaba virus. The inhibitory state in preinfected cells was shown to develop sequentially. Increase in the rate of deoxyribonucleic acid synthesis in the nuclei of preinfected cells occurred after infection with SV40. This rate of increase was significantly lower than that which occurred in SV40-infected cells which had not been preinfected. Cytosine arabinoside did not exert significant effect on the development of the inhibitory effect against SV40 in Yaba virus-infected cells.     

Induction of Epstein-Barr virus nuclear antigens.

Lymphocytes were infected with the QIMR-WIL strain of Epstein-Barr virus, and the induction of Epstein-Barr virus-associated nuclear antigens was determined by using the protein immunoblot. There was a temporal increase in six antigens, with Epstein-Barr nuclear antigen 2 being detected 1 day after infection. The appearance of these antigens was shown to be independent of cellular DNA synthesis.     

Effect of antisynaptosome antibodies on the metabolism of synaptosomal proteins

The authors studied the effect of intraventricular injection of antisynaptosomal gamma-globulin on the protein of synaptosomal proteins in the rat brain cortex. The effect was assesed according to the protein specific radioactivity 2 and 3 hours after injection into animals of 14C-protein hydrolysate of Chlorella. Injection of antisynaptosomal gamma-globulin provoked an increase and reduction in the specific radioactivity of the proteins under study 2 and 3 hours, respectively, after labeled precursor injection. It is assumed that interaction of synaptosomal antibodies with protein synaptosomal antigens produces intense decay of antigens, leading to activation of their synthesis.     

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.     

Physiological State of Human Embryonic Lung Cells Affects Their Response to Human Cytomegalovirus

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.     

Isolation and preliminary characterization of temperature-sensitive mutants of measles virus.

Twenty-four genetically stable temperature-sensitive mutants of measles virus were isolated after mutangenesis by 5-azacytidine, 5 fluorouracil, or proflavine. The restricted replication of all mutants at 39 C was blocked subsequent to cell penetration and could not be attributed to heat inactivation of virus infectivity. Complementation analysis was made possible through the use of poly-L-ornithine. The members of one complementation group exhibited wild-type RNA synthesis at the nonpermissive temperature and induced the synthesis of virus antigens. These mutants were found defective in both hemolysin antigen synthesis and cell fusion "from within," supporting the unitary hypothesis for these functions. The members of the other two complementation groups synthesized neither virion RNA nor detectable virus antigens at the nonpermissive temperature.     

Effect of Dibutyryl Cyclic AMP on the Induction of Epstein-Barr Virus in Hybrid Cells

Treatment of Epstein-Barr virus (EBV) negative somatic cell hybrids with 5'-iododeoxyuridine (IUdR) induced synthesis of EBV antigens and virus particles. When dibutyryl cAMP (Bt(2)-cAMP) was present in medium after exposure of cultures to IUdR, the incidence of cells synthesizing EBV early and virus capsid antigens was increased. The time necessary for appearance of EBV particles after induction by IUdR was significantly reduced in the presence of Bt(2)-cAMP. This enhancement was evident to a lesser degree with 3':5' cAMP than with Bt(2)-cAMP and did not occur with any other of the related compounds tested. The response observed was dose dependent. Untreated (no IUdR) EBV negative hybrid cells exposed to Bt(2)-cAMP also synthesized EBV antigens. The concentration of intracellular cAMP may act as one of the control mechanisms selecting for gene expression in this system.     

Requirements for the association of adenovirus type 2 E3/19K wild-type and mutant proteins with HLA antigens

The E3/19K protein of human adenovirus type 2 is a resident of the endoplasmic reticulum (ER). Immediately after synthesis, it associates with major histocompatibility complex class I antigens and prevents their intracellular transport and cell surface expression. We have generated several C-terminal deletion mutants of the E3/19K protein that are preterminated at various positions on both sides of the membrane-spanning segment of the protein. One of these mutants is terminated at the luminal side of the membrane (M310), and two are terminated in the hydrophobic segment (M374 and M392), whereas mutant M621 is terminated on the cytoplasmic side of the ER membrane. The M310, M374, and M392 mutants are soluble proteins. They do not associate with HLA antigens in transfected 293 cells, and they are, to some extent, secreted into the medium. The M621 mutant protein is integrated in the ER membrane, associates immediately after its synthesis with HLA antigens, and exits from the ER. By using either an in vitro translation system supplemented with microsomes or overexpression in insect cells, we showed that M374 and E3/19K are able to associate with HLA antigens. These results indicate that the conformation of the luminal part of the E3/19K protein is not grossly altered by the mutations. Rapid transport of the M374 mutant out of the ER and partial degradation of this protein may prevent the interaction with HLA class I antigens in transfected 293 cells.     

Effect of arginine deficiency on the reproduction of human cytomegalovirus.

In arginine-deprived human embryonic fibroblasts the reproduction cycle of human cytomegalovirus (CMV) is incomplete. Infectious virus cannot be demonstrated in cell disintergrates, and from among the CMV antigens only the diffuse cytoplasmic antigen is detectable by immunofluorescence. The antigens localized in the cell membrane and those appearing during the complete cycle as large granules or inclusion-like bodies in the nucleus do not appear in the absence of arginine. The CMV genome persists in the arginine-deprived culture; after re-feeding with arginine-containing medium, maturation of virions soon ensues. Maturation could be prevented by inhibitors of protein synthesis, but not by DNA inhibitors, added simultaneously with completion of the medium.     

Nature of the antigenic complex recognized by T lymphocytes. VII. Evidence for an association between TNP-conjugated macrophage membrane components and Ia antigens.

In the present study we examined the effects of anti-sera directed against guinea pig Ia antigens on the ability of TNP-conjugated macrophages to stimulate TNP-specific T lymphocyte proliferation. Treatment of macrophages with anti-Ia sera for 1 hr before, 1 hr immediately after, or as late as 24 hr after TNP-modification resulted in a reduced ability to stimulate the TNP-specific T cell. The inhibition produced by anti-Ia sera was specific and did not result from interference with the ability of macrophages to process TNP-conjugated membrane antigens in a nonspecific manner. Brief treatment with anti-Ia serum did not result in inhibition of Ia-antigen synthesis nor could evidence of carry-over of anti-Ia antibody into the lymphocyte cultures be obtained. These results demonstrate that anti-Ia sera interfere with the development of a TNP-specific immunogen on the macrophage surface and strongly suggest that an association exists between TNP-modified membrane proteins and Ia antigens on the macrophage surface.     

Cytomegalovirus-induced membrane antigens in productively infected cells.

Adsorbed but not penetrated virus can be removed from the CMV-infected cell membrane by digestion with cystine-activated papain. Membrane antigens appear on 80-90% of the infected cells 14-20 hr after infection as a result of de novo protein synthesis. Antigen synthesis can be blocked with inhibitors of protein synthesis, but not with DNA inhibitors. In the early stage of infection, pooled human convalescent serum reacted well with the membrane antigen, whereas pooled antiserum of rabbits immunized with CMV virion suspension gave a positive reaction with a small proportion of the cells. After the 48th hr, both the human and the rabbit serum pool reacted with the membrane of the infected cells. Absorption with cell cultured for 24 hr after CMV infection reduced the neutralization titres of the antisera only slightly but the titre reduction was considerable when absorption was performed with cells cultured for more than 48 hr after infection. It is concluded that on the membrane of cells productively infected by CMV at least two membrane antigens are present, one coded for by the DNA of the parent virus and another which is the product of the DNA of the virus progeny. The two antigens can be differentiated serologically.     

Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.     

Regulation of macrophage populations. II. Synthesis and expression of Ia antigens by peritoneal exudate macrophages is a transient event

We have evaluated the biosynthesis and surface expression of I-A antigens by peritoneal macrophages and found that both events terminated during the 1st day in culture, in contrast to the undiminished synthesis and expression of H-2K antigens. This pattern was observed regardless of the means by which the macrophages were elicited, but was subject to modulation for a limited period of time in vitro: phagocytic stimuli were able to augment both I-A synthesis and expression. The loss of I-A and the re-expression after phagocytosis were both reflected in the stimulatory capacity of these macrophages in the mixed leukocyte reaction. Moreover, we found that I-A-bearing macrophages were lost from the exudate in vivo after irradiation. Our data suggest that, as in vitro, this phenomenon is due to the transition of individual macrophages from I-A-positive to I-A-negative, and that constant renewal is required to maintain the I-A-bearing subset in vivo.     

Mechanisms of expression of herpes simplex virus-common surface antigens in clonal cells of a herpes simplex virus type 2-transformed line.

Rabbit antiserum hyperimmune to herpes simplex virus type 1 was used to study the expression of herpes simplex virus type-common surface antigens (CSA) by indirect immunofluorescence tests in three representative cell clones isolated from a herpes simplex virus type 2-transformed hamster line, 155-4. These three clones showed different phenotypes with respect to CSA expression: (i) a CSA-positive type (clone (155-4-213), in which the antigens increased soon (5 h) after seeding at 37 degrees C, but not after treatment with actinomycin D; (ii) a CSA-inducible type (clone 155-4-03), in which the antigens increased after treatment with actinomycin D (2 micrograms/ml) for 20 h, but not after seeding only; and (iii) a CSA-negative type (clone 155-4-16), in which the antigens did not increase after seeding or after actinomycin D treatment. CSA expression in the CSA-positive type was inhibited by 2-deoxy-D-glucose, but not by puromycin, suggesting that the expression required glycosylation, but not active protein synthesis. CSA expression in this type was insensitive to the protease inhibitors antipain and p-nitrophenyl-p'-guanidinobenzoate. On the other hand, actinomycin D-induced CSA expression in the CSA-inducible type was inhibited by both 2-deoxy-D-glucose and puromycin, suggesting that the induced expression required both glycosylation and protein synthesis. CSA expression induced in this type was sensitive to the two protease inhibitors at concentrations having little effect on overall cellular metabolism or cell viability. These results indicate that CSA expressions in the CSA-positive type and the CSA-inducible type are enhanced by different mechanisms.     

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.     

Mitogenic activity of the antigens isolated from different Staphylococcus aureus strains in relation to mouse and guinea pig splenocytes

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.     

Differential accumulation of oocyte nuclear proteins by embryonic nuclei of Xenopus

Oocyte nuclear proteins of Xenopus are distributed into the cytoplasm of the maturing egg after germinal vesicle breakdown. Later they are found in all cell nuclei of the embryo. At early stages of development, different nuclear proteins behave differently. A class of 'early shifting' antigens is accumulated by pronuclei and cleavage nuclei, whereas others appear to be excluded from the nuclei at early stages but are shifted into the nuclei at blastula or during and after gastrulation. Accumulation of 'late-shifting' nuclear antigens is a gradual process and occurs during a period characteristic of each protein. Multiple artificial pronuclei can be formed after injection of sperm nuclei, erythrocyte nuclei or pure lambda-DNA into unfertilized eggs. The artificial pronuclei accumulate early- but not late-shifting proteins. Early-migrating proteins rapidly accumulate into the germinal vesicle after de novo synthesis in the oocyte, indicating that the efficiency of translocation into nuclei is an intrinsic property of each protein. Artificial extension of the length of the cell cycle before midblastula transition does not lead to accumulation of the late-shifting nuclear antigens investigated.     

Antigen biosynthesis during the limitation of Yersinia pestis growth

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.