Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins.

Human T-cell lymphotropic virus type I (HTLV-I) double-spliced mRNA exhibits two GUG and two CUG codons upstream to, and in frame with, the sequences encoding Rex and Tax regulatory proteins, respectively. To verify whether these GUG and CUG codons could be used as additional initiation codons of translation, two chimeric constructs were built for directing the synthesis of either Rex-CAT or Tax-CAT fusion proteins. In both cases, the CAT reporter sequence was inserted after the Tax AUG codon and in frame with either the Rex or Tax AUG codon. Under transient expression of these constructs, other proteins of higher molecular mass were synthesized in addition to the expected Rex-CAT and Tax-CAT proteins. The potential non-AUG initiation codons were exchanged for either an AUG codon or a non-initiation codon. This allowed us to demonstrate that the two GUG codons in frame with the Rex coding sequence, and only the second CUG in frame with the Tax coding sequence, were used as additional initiation codons. In HTLV-I infected cells, two Rex and one Tax additional proteins were detected that exhibited molecular mass compatible with the use of the two GUG and the second CUG as additional initiation codons of translation. Comparison of the HTLV-I proviral DNA sequence with that of other HTLV-related retroviruses revealed a striking conservation of the three non-AUG initiation codons, strongly suggesting their use for the synthesis of additional Rex and Tax proteins.

Persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1.

Because synthesis of rRNA persists late during herpes simplex type 1 infection and because S6 phosphorylation is always correlated with efficient translation of ribosomal protein mRNA, we tested the hypothesis that ribosomal protein synthesis and ribosome biogenesis could persist after infection. At different times after infection, proteins were labelled with 35S for 1 h before harvesting and ribosomes were purified. Measurement of radioactivity incorporated into individual ribosomal proteins separated by two-dimensional PAGE demonstrated that ribosomal proteins are still synthesized and assembled into mature ribosomes up to late times during infection, while synthesis of beta-actin is severely inhibited. During expression of late genes, ribosome biogenesis was estimated to be 58% of that of the control as judged by [3H]uridine incorporation into rRNA. As for beta-actin mRNA, the level of ribosomal protein mRNA decreased progressively from the beginning of infection, reaching about 30% of the control level during expression of late genes. Taken together, these results demonstrate that ribosomal proteins are still synthesized up to the late time of infection and efficiently assembled into mature ribosomes, while there is a severe shutoff of the synthesis of other cellular proteins.

Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation.

To examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the alpha-subunit of CaMKII (alpha CaMKII) equivalent to mammalian neurons. The expression of the transfected alpha CaMKII gene or the endogenous beta CaMKII gene was monitored by RNase protection assays, and alpha CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of alpha CaMKII mRNA and alpha CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from alpha CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing alpha CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the alpha CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of alpha CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that alpha CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.

Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element.

The DNA sequence coding for the 5' untranslated region of herpes simplex virus type 1 ICP22 mRNA mediates a high level of gene expression.

The sequence coding for the 5' untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5'UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5'UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5'UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5'UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5'UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.

Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction.

A strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co-amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co-migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.

The herpes simplex virus type 1 US11 gene product is a phosphorylated protein found to be non-specifically associated with both ribosomal subunits.

Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to probe Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.

Herpes simplex virus type-1 immediate-early gene expression and shut off of host protein synthesis are inhibited in neomycin-treated human epidermoid carcinoma 2 cells.

Infection of human epidermoid carcinoma-2 (HEp-2) cells by Herpes simplex virus type 1 (HSV-1) leads to significant activation of inositol phospholipid turnover after 15 min. The effect of neomycin, an inhibitor of inositol phospholipid turnover, has been investigated for its effect on HSV-1 multiplication in HEp-2 cells. HSV-1 multiplication is inhibited by neomycin. This inhibition is not due to a block of virus adsorption or penetration. Neomycin inhibits the expression of virus immediate-early genes, as well as expression of early genes and viral DNA synthesis. In neomycin-treated cells, the usual virion-associated shut off of host protein synthesis does not occur. These results indicate that the inositol phospholipid pathway is involved in immediate-early gene expression and shut off of host protein synthesis in HEp-2 cells.

Herpes simplex virus type-1-induced stimulation of ribosomal protein S6 phosphorylation is inhibited in neomycin-treated human epidermoid carcinoma 2 cells and in ras-transformed cells.

Neomycin, an inhibitor of inositol phospholipid turnover, prevents Herpes-simplex-virus-type-1 (HSV-1)-induced stimulation of ribosomal protein S6 phosphorylation, but does not impair the S6 phosphorylation induced by serum. Long-term treatment with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C activity, does not inhibit virus-induced S6 phosphorylation. In ras-transformed cells, S6 phosphorylation is not stimulated after HSV-1 infection. These results suggest that activation of the inositol phospholipid pathway is involved in the HSV-1-induced stimulation of S6 phosphorylation. However, protein kinase C activation does not appear to be necessary for HSV-1-induced S6 phosphorylation.

Ribosome and protein synthesis modifications after infection of human epidermoid carcinoma cells with herpes simplex virus type 1.

Modifications of ribosomes have been investigated in human epidermoid carcinoma-2 cells at different stages of herpes simplex virus type 1 infection. Very early in infection, there is an increase in ribosomal protein S6 phosphorylation even in the absence of serum. The same result is obtained in the presence of actinomycin D. At early infection time, ribosomal proteins S2, S3a and Sa are newly phosphorylated. At early and early-late times, three phosphorylated non-ribosomal proteins (v1, v2 and v3) are differently associated temporally to ribosomes. Analyses of proteins extracted from 40S subunits, 80S ribosomes and polysomes show that v1 and v2 are distributed differently among the different ribosomal populations. S6 phosphopeptides were found to be identical after serum stimulation and after viral infection. In every case phosphoserine and phosphothreonine were identified in S6. Only phosphoserine was found in other phosphorylated proteins. Our results indicate that herpes simplex virus type 1 is able to modify pre-existing ribosomes: (i) by stimulating a pre-existing kinase for S6 phosphorylation even in the absence of serum and of viral genome expression; (ii) by inducing new specific kinase activity(ies); and (iii) by association of new, phosphorylated proteins to ribosomes. These ribosomal modifications are correlated with changes in protein synthesis, as shown by two-dimensional electrophoretic analyses of newly synthesized 35S-labelled proteins.

Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus.

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock-infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the same mono-phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.

Location and cloning of the herpes simplex virus type 2 thymidine kinase gene.

The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.