The interferon-induced double-stranded RNA-activated human p68 protein kinase inhibits the replication of vaccinia virus

The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (p68 kinase) has long been implicated as one of the antiviral agents responsible for overcoming virus infections. To investigate the antiviral potential of p68 kinase, we have generated a recombinant vaccinia virus that expresses human p68 kinase under the control of lac operator/repressor element. Upon induction of p68 kinase gene with the inducer isopropyl-beta-D-thiogalactoside (IPTG), we observed in cultured cells a severe (> 90%) inhibition of virus protein synthesis; this inhibition correlated with autophosphorylation of p68 kinase. As a result of inhibition in the synthesis of virus polypeptides, there was a 100-fold decrease in virus yields. When cells were infected with the recombinant virus expressing lys296-->arg296 mutant p68 kinase there was no reduction in virus yields. Our findings demonstrate that human p68 kinase once activated severely inhibits vaccinia virus replication as a result of inhibition of protein synthesis.     

Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1

Cells respond to environmental stress and proinflammatory cytokines by stimulating the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase cascades. Infection of eukaryotic cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h post-infection. By using a series of mutant viruses, we showed that the virion transactivator protein VP16 stimulates p38/JNK, whereas no immediate-early, early, or late viral expressed gene is involved. We identified the stress-activated protein kinase kinase 1 as an upstream activator of p38/JNK, and we demonstrated that activation of AP-1 binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in the cellular levels of Jun protein. We suggest that activation of the stress pathways by HSV-1 infection either represents a cascade triggered by the virus to facilitate the lytic cycle or a defense mechanism of the host cell against virus invasion.     

Interferon regulatory factor 1 is induced by interferon-gamma equally in neurons and glial cells

Class I MHC protein is induced in glia but not mature neurons by IFN-gamma. We have compared IFN-gamma signal transduction in these populations. There were identical levels of STAT1 homodimers and IRF-1 by gel-shift and IRF-1 mRNA was induced equally. However class I MHC, beta2-microglobulin and interleukin 1-beta converting enzyme mRNA levels were greatly reduced in neurons. These experiments show that there is no defect in expression of IRF-1 in response to IFN-gamma in mature mouse neurons but that insufficient class I MHC gene expression is induced for detectable cell surface protein expression.     

Interferon (IFN) consensus sequence-binding protein, a transcription factor of the IFN regulatory factor family, regulates immune responses in vivo through control of interleukin 12 expression

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. To gain a mechanistic understanding of the contributions of ICSBP to humoral and cellular immunity, we characterized the responses of control and ICSBP-/- mice to infection with influenza A (flu) and Leishmania major (L. major). Mice of both genotypes survived infections with flu, but differed markedly in the isotype distribution of antiflu antibodies. In sera of normal mice, immunoglobulin (Ig)G2a antibodies were dominant over IgG1 antibodies, a pattern indicative of a T helper cell type 1 (Th1)-driven response. In sera of ICSBP-/- mice, however, IgG1 antibodies dominated over IgG2a antibodies, a pattern indicative of a Th2-driven response. The dominance of IgG1 and IgE over IgG2a was detected in the sera of uninfected mice as well. A seeming Th2 bias of ICSBP-deficient mice was also uncovered in their inability to control infection with L. major, where resistance is known to be dependent on IL-12 and IFN-gamma as components of a Th1 response. Infected ICSBP-deficient mice developed fulminant, disseminated leishmaniasis as a result of failure to mount a Th1-mediated curative response, although T cells remained capable of secreting IFN-gamma and macrophages of producing nitric oxide. Compromised Th1 differentiation in ICSBP-/- mice could not be attributed to hyporesponsiveness of CD4(+) T cells to interleukin (IL)-12; however, the ability of uninfected and infected ICSBP-deficient mice to produce IL-12 was markedly impaired. This indicates that ICSBP is a deciding factor in Th responses governing humoral and cellular immunity through its role in regulating IL-12 expression.     

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.