The latent form of the dimeric transcription factor NF-kappa B is sequestered in the cytoplasm by proteins containing ankyrin repeats, such as 1 kappa B alpha and beta, or by the p105 precursor form of the NF-kappa B p50 subunit. Tumor necrosis factor alpha or virus infection can cause targeted destruction of 1 kappa B and nuclear translocation of NF-kappa B. Following translocation, NF-kappa B mediates immune, inflammatory, or anti-apoptotic responses. Here we present evidence that beginning at around 6 h postinfection, herpes simplex virus (HSV) induces a persistent translocation of NF-kappa B into the nucleus of C33 cells, coincident with loss of both 1 kappa B alpha and 1 kappa B beta. Translocation failed to occur when infecting virus was preincubated with neutralizing antibody to viral envelope glycoproteins gD or gH, thus preventing entry, or when cells infected with viruses expressing mutated forms of immediate-early regulatory proteins lCP4 or lCP27. Surprisingly, no increase in the trans-activation function of NF-kappa B, as assayed by transient expression of CAT, was detected following HSV infection. The significance of NF-kappa B nuclear translocation for virus replication was demonstrated by an 80-90% reduction in virus yield following infection of C33 cells expressing a constitutive repressor form of 1 kappa B alpha. Models that reconcile nuclear translocation of NF-kappa B with the inability to detect NF-kappa B-dependent gene expression are discussed.

Studies of the size, composition, and structure of the deoxyribonucleic acid (DNA) of the F and G prototypes of herpes simplex virus (HSV) subtypes 1 and 2 (HSV-1 and HSV-2) showed the following. (i) As previously reported by Goodheart et al. HSV-1 and HSV-2 DNA have a buoyant density of 1.726 and 1.728 g/cm3, corresponding to 67 and 69 guanine i cytosine moles per cent, respectively. The difference in guanine plus cytosine content of the DNA species was confirmed by the finding of a 1 C difference in Tm. (ii) The DNA from purified virus on cocentrifugation with T4 DNA in neutral sucrose density gradients sedimented at 55S, corresponding to 99 ± 5 million daltons in molecular weight. HSV-1 and HSV-2 DNA could not be differentiated with respect to size. (iii) Cosedimentation of alkali-denatured DNA from purified virus with T4 DNA on alkaline sucrose density gradients consistently yielded several bands of single-stranded HSV DNA ranging from fragments 7 x 106 daltons to intact strands 48 X 106 daltons in molecular weight. Naturally occurring herpes simplex viruses (HSV) differ in a number of biological, physical, and immunological properties (5, 7, 8, 10, 17-19, 20, 27). As determined in neutralization tests, most [but not all (31)] fresh isolates fall into two subtype groups designated as 1 (HSV-1) and 2 (HSV-2). This report concerns the properties of HSV deoxyribonucleic acid (DNA). Previous reports have estimated the molecular weight of HSV-1 DNA to be 68 x 106 (24) to 100 x 106 daltons (2) and to contain 65 to 68 per cent guanine plus cytosine (10, 11, 20,22,23). HSV-2 DNA was reported (10) to contain 70.4 moles per cent guanine plus cytosine, and nothing was known of its size or relatedness to HSV-1 DNA. In the experiments described in this paper, we have directly compared the size, composition, and structure of HSV-1 and HSV-2 DNA. MATERIALS AND METHODS Solutions. Virus buffer consisted of 0.15 M NaCl and 0.02 M tris(hydroxymethyl)aminomethane (Tris)hydrochloride, pH 7.5. Neutral DNA buffer consisted of 1 M NaCl, 0.001 M ethylenediaminetetraacetic acid (EDTA), and 0.05 M Tris-hydrochloride, pH 7.5. Alkaline DNA buffer consisted of 0.8 M NaCl, 0.3 M NaOH, and 0.001 M EDTA. Reticulocyte standard buffer (RSB) consisted of 0.01 M sodium chloride, 0.001 M magnesium chloride, 0.01 M Tris, pH 7.5. Standard saline citrate (1 X SSC) consisted of 0.15 M sodium chloride, 0.015 M sodium citrate. SDS buffer consisted of 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris,

Each poly(A) containing cytoplasmic AD-2 MRNA contains at its 5' terminus the general structure m7 GpppN1 pN2p or m7 GpppN1mpN2mpNp as well as an average of 4 m6A and 0.5-1 m5C residues per molecule. Almost all of the N1m residues are adenine derivatives including Am, m6Am and probably m26,6Am. The N2m is mostly Cm but small amounts of the other three methylated bases are also present. All the methylated constitutents of mRNA are distant from the 3' terminal poly(A). The amount of m6A appears to be greater in larger mRNA than in smaller mRNA. Nuclear Ad-2 specific RNA also contains caps, m6A, and m5C with about twice as much m6A relative to caps as cytoplasmic mRNA. The similarity of Ad-2 nuclear and mRNA to HeLa hnRNA and mRNA suggests that adenovirus mRNA production is a good model for eukaryotic mRNA production.

Cells activate the transcription factor NFB in a wide variety of situations, including responses to stress-inducing insults such as UV irradiation and virus infection or in response to cytokines such as tumor necrosis factor alpha (TNF-␣). NFB has an important role in suppression of apoptosis and regulates the expression of many important antiapoptotic functions (5,31,48,50). NFB, as a p65/p50 heterodimer, is normally sequestered in the cytoplasm in a complex with inhibitor of B (IB). IB␣ is targeted for phosphorylation at serine residues 32 and 36, and IB␤ is targeted for phosphorylation at serine residues 19 and 23 (42, 46, 51), by the multisubunit IB kinase (IKK) (11,23,36,52,58). This phosphorylation triggers its polyubiquitylation and destruction by the 26S proteosome (6,7,12), and, as a result, NFB is translocated to the nucleus (14, 57). Key roles for IKK and IB in NFB signaling were demonstrated in studies in which overexpression of a kinase-dead, trans-dominant form of IKK prevents IB phosphorylation and inhibits NFB activation (36, 52). IKK is activated by phosphorylation mediated by mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) MEKK1, -2, or -3 or NFBinducing kinase (NIK) (23,30,37,59). Under stress conditions, the double-stranded RNA-activated protein kinase (PKR) has been shown to activate NFB through a pathway dependent on NIK and IKK (57). Distinct roles for the catalytic components of the IKK have been recognized. IKK␣ appears to play a major role in transducing signals for NFB activation during embryonic development (18, 45), while IKK␤ is essential for cytokine and other stress-induced signaling pathways (10,27,29). Besides cytoplasmic roles in the activation of NFB, recent studies have identified IKK␣ and the IKK scaffold protein IKK␥/NEMO in direct regulation of NFB-dependent transcription in the nucleus (2, 49, 53). NFB activation is also dependent on distinct signaling pathways which target p65 for phosphorylation (32).The ability of herpes simplex virus type 1 (HSV-1) to activate NFB has been well documented (1,15,40). Beginning at 3 to 5 h postinfection (hpi), HSV-1 induced a strong and persistent nuclear translocation, increased p50/p65-dependent DNA binding activity as measured by electrophoretic mobility shift assay (EMSA), and induced activation of a 3XNFB-luciferase reporter. Persistent NFB activation required virus binding and entry as well as de novo infected cell protein synthesis, including expression of functional viral immediateearly (IE) protein ICP27. Activation was also accompanied by increased IKK activity and loss of both IB␣ and IB␤. Interference with NFB activation occurred following expression of a dominant-negative IB␣ (DNIB) containing alanine substitutions for serine residues 32 and 36 normally targeted by IKK. The resulting substantial reduction in NFB EMSA activity correlated with a reduction in virus yield. The latter may be related to the reported role of NFB in preventing HSV-1-induced apoptosis (15). The foregoing results argue that the observe...

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G(0)-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein-E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G(1) from proceeding through the restriction point and for cells in S from completing a round of DNA replication.