The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (alpha4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled US3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type alpha4 gene but from which the US3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the US3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the US3 gene as the principal viral product required to block apoptosis.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Cells infected with herpes simplex virus 1 (HSV-1) undergo productive or latent infection without exhibiting features characteristic of apoptosis. In this report, we show that HSV-1 induces apoptosis but has evolved a function that blocks apoptosis induced by infection as well as by other means. Specifically, (i) Vero cells infected with a HSV-1 mutant deleted in the regulatory gene alpha 4 (that encodes repressor and transactivating functions), but not those infected with wild-type HSV-1(F), exhibit cytoplasmic blebbing, chromatin condensation, and fragmented DNA detected as a ladder in agarose gels or by labeling free DNA ends with terminal transferase; (ii) Vero cells infected with wild-type HSV-1(F) or cells expressing the alpha 4 gene and infected with the alpha 4- virus did not exhibit apoptosis; (iii) fragmentation of cellular DNA was observed in Vero cells that were mock-infected or infected with the alpha 4- virus and maintained at 39.5 degrees C, but not in cells infected with wild-type virus and maintained at the same temperature. Wild-type strains of HSV-1 with limited extrahuman passages, such as HSV-1 (F), carry a temperature-sensitive lesion in the alpha 4 gene and at 39.5 degrees C only alpha genes are expressed. These results indicate that the product of the alpha 4 gene is able to suppress apoptosis induced by the virus as well by other means.

Infected cell protein no. 4 (ICP4), the major regulatory protein encoded by the alpha 4 gene of herpes simplex virus 1, binds to a site (alpha 4-2) at the transcription initiation site of the alpha 4 gene. An earlier report described the construction of recombinant viruses that contained chimeric genes (alpha 4-tk) that consisted of the 5' untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene and showed that disruption of the alpha 4-2 binding site by mutagenesis derepressed transcription of this gene (N. Michael and B. Roizman, Proc. Natl. Acad. Sci. USA 90:2286-2290, 1993). This experimental design was used to determine the effect of displacement of the alpha 4-2 binding site on the repression of alpha 4 gene transcription by ICP4. We report the following findings. (i) In the absence of the alpha 4-2 binding site, at 4 h after infection, alpha 4-tk RNA levels increased 10-fold relative to the corresponding RNA levels of a gene that contained the alpha 4-2 site at its natural location. Displacement of the alpha 4-2 binding site by approximately one, two, and three turns of the DNA helix, i.e., by 10, 21, and 30 nucleotides downstream of the original site, increased the concentration of alpha 4-tk RNA 2.4-, 3.5-, and 5.8-fold, respectively. (ii) Displacement of 16 nucleotides, i.e., approximately 1.5 helical turns, increased the accumulation of alpha 4-tk by 5.3-fold, i.e., more than predicted by displacement alone. (iii) At 8 h after infection in the absence of the binding site, the accumulation of alpha 4-tk RNA increased 13.6-fold. However, in cells infected with recombinants that carried displaced alpha 4-2 binding sites, RNA accumulation decreased relative to the levels seen at 4 h after infection. The insertion of DNA sequences in order to displace the alpha 4-2 binding site had no effect on accumulation of RNA in the presence of cycloheximide, i.e., in the absence of ICP4, or on maximum accumulation of alpha 4-tk RNA in the absence of the alpha 4-2 binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied the effect of measles virus (MV) infection on the expression of MHC class II molecules and on the antigen-presenting function in human peripheral blood monocytes in vitro. The expression of HLA-DR. -DQ, and -DP molecules was up to 10-fold higher in MV-infected than that in uninfected monocytes. This effect did not change upon stimulation of monocytes with LPS, and was not mediated by IFNγ. In contrast to results obtained with peripheral monocytes. MV infection in a human monocytic cell line. THP-1, did not enhance the basic MHC class II expression, but strongly inhibited the IFN-γ-induced expression. High levels of MHC class II expression in MV-infected monocytes were associated with capacity to present MV antigens, but the capacity to present unrelated antigens was strongly reduced. These results indicate how MV infection in monocytes may suppress the antigen-medicated T cell proliferation, still allowing the expansion of MV-specific T cell clones.

The genomic 3' noncoding region (NCR) of nonsegmented negative-strand RNA viruses contains recognition site(s) for the polymerase complex, while the RNA plus-strand leader sequence (LS) is probably involved in RNA encapsidation. It is known that host-encoded factors play a role in transcription and replication of some of this group of viruses. Here we report that cellular proteins interact with the genomic 3' NCR and with the plus-strand LS RNA of an important human pathogen, measles virus (MV), a member of the family Paramyxoviridae. Using gel retardation assay and RNA footprinting analysis, we demonstrated that in Vero cells, host-encoded proteins bind specifically to domains within these two sequences. A polypeptide of about 20 kDa binding to the 3' NCR and two polypeptides of about 22 and 30 kDa interacting with plus-strand LS were detected by RNA-protein UV cross-linking. Different RNA-binding activities were found in cells differing in permissiveness to MV replication. The results suggest a role for host-encoded proteins in MV replication.

Monocytes may play a role in the immunologic abnormalities caused by measles. The effect of measles virus (MV) infection on peripheral blood monocyte functions is poorly known. We report that MV-infected PBM have an altered pattern of IL-1 beta and TNF-alpha production in response to stimulation with LPS and PMA in vitro. MV-infected peripheral blood monocytes produced higher amounts of IL-1 beta, whereas the production of TNF-alpha was reduced. The same effect was observed in the human monocytic cell line THP-1, which was used for RNA analysis. An increased steady-state level of IL-1 beta mRNA was observed in MV-infected cells, and the level of TNF-alpha mRNA was reduced. However, both IL-1 beta and TNF-alpha had about 50% increased transcription rate. Analysis of the mRNA stability after transcriptional block by actinomycin D showed that the TNF-alpha mRNA had a reduced half-life in MV-infected cells (about 30 vs 80 min in uninfected cells), whereas IL-1 beta mRNA stability was similar in uninfected and MV-infected cells. These results indicate that MV infection disturbs the immunoregulatory network by interfering with the monocyte functions.

We analyzed the effect of exogenous human leukocyte interferon (IFN)-alpha on measles virus (MV) replication in human peripheral blood mononuclear cells (PBMC). The release of infectious virus was progressively reduced by increasing concentrations of IFN-alpha, and blocked with an IFN-alpha concentration of 1000 U/ml. In order to detect a possible target of this inhibitory effect, viral transcription and translation events were analyzed. The synthesis of MV mRNAs was reduced, but not blocked, in the presence of IFN-alpha. However, this effect was not specific on the viral RNAs, but due to a general inhibition of RNA synthesis in IFN-treated PBMC. The expression of viral polypeptides was also inhibited in a dose-dependent manner by exogenous IFN-alpha, but a low level of protein synthesis was detected by both Western blotting and immunofluorescence techniques, even with the maximum amount of IFN-alpha used (1000 U/ml). These findings account for a partial maintenance of the viral replicative cycle, even when the production of infectious virus is blocked. Moreover, the effect of IFN-alpha is not specifically targeted on the virus macromolecular synthesis.