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Background: Patients treated with lithium salts for manic depression had a lower incidence of herpes simplex infections. Initial studies in our laboratory demonstrated that addition of LiCl in cultures of human endothelial cells infected with herpes simplex virus suppressed viral replication and allowed synthesis of host proteins., Experimental Design: Based on the above observations, we decided to study the optimal condition for the lithium effect and determine the process of inhibition of viral replication. Endothelial cell cultures infected with herpes simplex virus-1 were exposed to LiCl at various times postinfection. The levels of host and viral mRNAs were measured by Northern and slot blot hybridization. The pattern of protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot and replication was assessed by plaque assay., Results: LiCl inhibited virus replication in a dose- and time-dependent manner as was reflected in the sharp decrease or absence of infectious virus production. The condition for optimal effects of LiCl were the addition of the salt between 0-3 hours postinfection, and at a concentration of 30 mM. LiCl suppressed the synthesis of viral polypeptides, whereas the synthesis of host proteins was maintained. Similar results were observed with phosphonoacetic acid, an inhibitor of viral DNA polymerase. NaCl, at the same concentration as LiCl, did not prevent the virus-induced inhibition of host cell protein synthesis. The level of host mRNA for fibronectin, thrombospondin, collagen type IV, actin, and plasminogen activator inhibitor-1 were maintained in the presence of LiCl. mRNAs for viral proteins, ICP-4 and DNA polymerase were nearly undetectable when LiCl was added with the virus (0 time postinfection)., Conclusions: The data indicate that LiCl treatment results in suppression of herpes virus mRNAs, i.e., mRNAs for ICP-4 and DNA polymerase, thereby inhibiting replication. On the other hand, the levels of host mRNAs are maintained to varying degrees depending on the message. The data suggest that a very early step in the process of viral replication is affected by LiCl, since the drug is maximally effective when added with the virus.

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.

Earlier studies from this laboratory have shown that infection of vascular cells with herpes simplex virus 1 or 2 (HSV-1, HSV-2) results in the differential suppression of extracellular matrix proteins including fibronectin (FN), type IV collagen, thrombospondin (TSP) and Factor VIII von Willebrand protein. The present study was designed to determine whether a correlation exists between suppression of synthesis of specific proteins and their mRNA levels. We have measured the steady-state levels of mRNAs for several extracellular matrix proteins (type IV collagen, FN and TSP) and two intracellular proteins (actin and tubulin) in human endothelial cells (EC) following HSV-1 infection. The results show that during the first 5 h post-infection, when there is a rapid decrease in the synthesis of extracellular matrix proteins, the steady-state levels of the corresponding mRNAs remain relatively high, but progressively decline to levels of less than 20% by 13 h post-infection. These findings suggest that in the early hours post-infection there is an alteration in the translatability of the hybridizable message followed by degradation in the later hours.

We compared the structure and subunit composition of laminin in less tumorigenic mouse A9 and highly tumorigenic mouse A9HT cells by pulse-chase studies. During a 15' pulse, the ratio of laminin B1 to B2 subunits in cell lysates is 1:1 in both the A9 and A9HT cells; however, after a 3 hr chase, this ratio changes to 6:1 and 2:1 in the A9 and A9HT cells, respectively. Analysis of mature laminin subunits in culture media after a 3 hr chase also showed a similar higher ratio of B1 to B2 in the A9 cells as compared to the A9HT cells. The higher ratio of B1 to B2 subunits in A9 cells was evident as early as after a 30' pulse. A comparative analysis of steady-state levels of mRNAs for the laminin subunits B1 and B2 between A9 and A9HT cells showed a ratio of 1:1 for B1 and a ratio of 1:1.65 for B2. The ratio of B1 to B2 mRNAs in A9 cells was 1:1.3 whereas in A9HT cells it was 1:2.5, suggesting changes in the processing of mRNA in the highly tumorigenic A9HT cells. These observations suggest that the processing of laminin B subunits is altered during the process of increased tumorigenicity, thus resulting in the synthesis and secretion of structurally different laminin in tumorigenic A9HT cells as compared to the parent and less tumorigenic A9 cells.

Herpes simplex virus type 1 produces an early decrease in protein synthesis in cultured smooth muscle cells isolated from human umbilical artery. To confirm that suppression of host protein synthesis occurs before virus protein expression, cultures were treated with actinomycin D (5 micrograms/ml) continuously from the beginning of infection. In the presence of actinomycin D, by 3.5 hours postinfection, virus-infected cultures showed much less incorporation of [35S]methionine into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than did identically treated mock-infected cultures. In the absence of actinomycin D, there was incorporation of radiolabel into viral proteins as early as 4.5 hours postinfection. Hybridization of slot-blotted total RNA demonstrated that virus-infected cultures treated with and without actinomycin D showed a marked decrease in hybridizable RNA by 2 hours PI when compared with mock-infected controls. This decrease was seen with cDNA probes for the mRNA for secreted extracellular matrix macromolecules (fibronectin, thrombospondin and collagen chains alpha 2(I), alpha 1(III] as well as with cDNA probes for RNA of intracellular macromolecules actin and beta-tubulin. In vitro translation of total RNA isolated from infected human smooth muscle cells at 2 and 5 hours postinfection resulted in far less [35S]methionine incorporation into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than would be predicted from the amount of hybridizable RNA available. The results suggest that host message is rapidly rendered nonfunctional as well as degraded by means of a virion-competent mechanism(s).

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.

Two human cDNA libraries prepared from normal fibroblast (GM3348) and rhabdomyosarcoma (CCL136) mRNAs were screened under cross hybridization conditions with a genomic fragment coding for exons 2 and 3 of the avian type III procollagen COOH-propeptide (Yamada, Y., Mudryj, M., Sullivan, M. and deCrombrugghe, B. (1983) J. Biol. Chem. 258, 2758-2761). One cDNA clone containing a 1.12 kb insert was isolated from the CCL136 library and later used to identify a GM3348 derived clone with a 2.4 kb insert. Comparison of the human and avian type III C-terminal propeptides revealed much more divergence in the first 54 amino acids following the terminal cysteine of the triple helical region than is present in the alpha 1(I) and alpha 2(I) procollagen chains of these species. Analysis of poly (A+) RNA from normal human fibroblast and tumor cell lines showed that they differed greatly in the relative amounts of alpha 1(I), alpha 2(I), and alpha 1(III) mRNAs. Furthermore, as we previously reported for the alpha 1(I) and alpha 2(I) transcripts, multiple mRNAs also hybridize to the cloned alpha 1(III) DNA.

Basement membrane collagen from bovine anterior lens capsules (ALC) was isolated by a non-degradative procedure and characterized. The material was obtained by extracting the ALC with 0.5 M acetic acid, passing the extract through a DEAE-cellulose column, collecting and concentrating the unbound fraction. Amino acid and carbohydrate analysis, polyacrylamide gel electrophoresis and rotary shadowing electron microscopy showed the purified extract to have the characteristics of basement membrane procollagen. Examination by rotary shadowing revealed the material to consist of type IV procollagen-like tetramers in various degrees of aggregation. When this purified procollagen was injected into rabbits, antibody of high titer and specificity was obtained. The competitive ELISA was then used to demonstrate lack of reactivity of the antibody with collagen types I, II, III and V and fibronectin. The electroimmunoblot technique was used to demonstrate lack of reactivity with laminin. Competition with collagenase resistant fragments of type IV procollagen representing the carboxyl terminal domain (NCI) and the 7-S domain, as well as with a pepsin-resistant alpha 1 (IV)125K chain was markedly weaker as compared to the native type IV procollagen molecule.

Screening of a human fibroblast cDNA library with a mouse type IV procollagen clone resulted in one 1.05-kilobase isolate that was used to identify a 1.7-kilobase clone overlapping the former by less than 150 nucleotides. EcoRII digestion revealed that the larger clone exhibited the pattern characteristic of DNA coding for a collagenous sequence. Blot hybridization to RNA from mouse F9 stem cells and from these cells treated with retinoic acid and N6, O2'-dibutyryl-cAMP showed induction of type IV mRNA. DNA sequencing and comparison of the derived amino acids with the reported protein data demonstrated that the clones encode part of the alpha 1 chain of human type IV procollagen. Alignment of alpha 1 (IV) with other human procollagens showed minimal but detectable homology. A small cluster of charged residues in the alpha chain is partially shared by type IV. In close proximity is an interruption in the alpha 1 (IV) Gly-Xaa-Yaa region corresponding to the 3' end of a unique proline-free, and therefore also less rigid, area in other collagen triple helices. Analysis of the carboxyl-terminal alpha 1 (IV) peptide showed a repeat symmetry possibly dictated by the six cysteines in each half of the structure. The position of five cysteines in addition to four tyrosine/tryptophan groups allowed a correlation to be drawn between the 3' noncollagenous type IV region and the larger, highly conserved carboxyl propeptides of other human procollagens. Such similarities in the different chains may define functional domains conserved throughout evolution.

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.

The DNA sequence corresponding to the 1.3 kb 3' untranslated region of the 6.5 kb human procollagen alpha 1(IV) mRNA was determined and compared with the mouse sequence obtained from 3' cDNA and genomic clones overlapping the reported 5' half (Oberbaumer et al., 1985, Eur. J. Biochem. 147:217). Although four AAUAAA hexanucleotides are found in the human and seven in the mouse RNAs, Northern blot hybridization showed almost exclusive utilization of the most 3' sequence, in contrast to the pattern seen when using alpha 1(I), alpha 2(I), alpha 1(III) and alpha 2(V) procollagen probes. Moreover, the ninety nucleotides 5' to the poly A tail in the major alpha 1(IV) mRNAs exhibit a much greater degree of interspecies homology than those encompassing the other three shared AAUAAA recognition signals. Further examination of this highly conserved area revealed the presence of two "consensus sequences" found in the 3' noncoding region of a number of RNA polymerase II transcribed genes (Mattaj and Zeller, 1983, Embo J. 2:1883) and, unexpectedly, some similarity with the nucleotides 5' to the poly A attachment signals in other procollagen mRNAs.