The ND10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 E4 intranuclear inclusion bodies in cultured keratinocytes and in warts

Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase.     

Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner

CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a-/-) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a-/- mice compared with WT mice. Similarly, Cd59a-/- T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.     

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.     

Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila

The gene for a male ejaculatory protein, Acp26Aa, in four sibling species of the Drosophila melanogaster subgroup has previously been shown to have a nonsynonymous rate (Ka) of nucleotide substitution that is indistinguishable from the synonymous rate (Ks). By examining this gene in two other species of this subgroup, we found that Ka is generally large and can sometimes be more than twice as large as Ks. This suggests that positive selection may be operating at this locus of male reproduction.      

Modulation of the level of expression of cellular genes in adenovirus 12-infected and transformed human cells.

The level of expression of thymidine kinase (TK), heat shock protein 70 (HSP70), beta-tubulin and p53 was assessed in human embryo kidney cells (HEKs) infected with adenovirus type 12 (Ad 12) and Ad 12 early region 1 (E1) mutants. HSP70, beta-tubulin and p53 levels were unchanged but TK activity was dramatically increased following wild-type infection. The initial activation of TK required the expression of the product of the E1A 13S mRNA but sustained expression only occurred with those viruses expressing the E1B proteins as well. A number of human cell lines transformed with either Ad 12 or Ad 5 E1 DNA were also assessed for the level of expression of HSP70, beta-tubulin and p53. Both HSP70 and beta-tubulin levels were greatly increased compared with primary human cells although there was considerable variation between lines. p53 was only expressed at high levels in Ad 12-transformed lines expressing E1A and E1B proteins.     

Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: independence from blood coagulation.

We have previously shown that a serum protein, termed differentiation reversal factor (DRF), is responsible for neurite retraction in differentiated cultures of an adenovirus 12 (Ad12) transformed human retinoblast cell line. Data is presented here to show that DRF is identical to the serine protease prothrombin. Both proteins have been immunoprecipitated using an antibody raised against purified prothrombin and have been shown to hydrolyse a specific thrombin substrate only after activation by the snake venom ecarin. Following addition to Ad12 HER 10 cells, which had previously been differentiated by culture in the presence of 2 mM dibutyryl cAMP in serum-free medium, thrombin and prothrombin caused half-maximal retraction of neurites at concentrations of 0.5 ng/ml and 20 ng/ml respectively. Interestingly, activation of prothrombin was shown to be unnecessary for biological activity. Using the inhibitor di-isopropylfluorophosphate (DIP), we have shown that abrogation of the proteolytic activity of thrombin also results in a loss (greater than 2000 fold) of differentiation reversal activity. Thrombin and its zymogen both stimulated the mitosis of differentiated Ad12 HER 10 cells to a similar extent. In addition, differentiation reversal was highly specific since, at physiologically significant concentrations, closely related serine proteases did not cause neurite retraction. Prothrombin and thrombin also reversed morphological differentiation in the SK-N-SH neuroblastoma cell line and in heterogeneous cultures of cells from various regions in the human foetal brain.     

The MHC E locus in macaques is polymorphic and is conserved between macaques and humans

Although the functions of the molecules encoded by the classical MHC class I loci are well defined, no function has been ascribed to the molecules encoded by the non-classical MHC class I loci. To investigate the evolution and conservation of the non-classical loci, we cloned and sequenced HLA-E homologues in macaques. We isolated four E locus alleles from five rhesus monkeys and two E locus alleles from one cynomolgus monkey, which indicated that the E locus in macaques is polymorphic. We also compared the rate of nucleotide substitution in the second intron of the macaque and human E locus alleles with that of exons two and three. The rate of nucleotide substitution was significantly higher in the introns, which suggested that the E locus has evolved under selective pressure. Additionally, comparison of the rates of synonymous and non-synonymous substitutions in the peptide binding region versus the remainder of the molecule suggested that the codons encoding the amino acids in the peptide binding region had been conserved in macaques and humans over the 36 million years since macaques and humans last shared a common ancestor.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers UO2976–UO2981     

FXII

The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, FXII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the FXII zymogen is converted to active enzymes. FXII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. FXII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein FXII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of FXII can be of benefit in certain clinical conditions.