Protein kinase PKR is activated during viral infection and phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, which were traced to positively-selected residues near the eIF2α-binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in non-human model systems.

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.

Metastability of the native form of proteins has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus and the strained native structure of serpins (serine protease inhibitors) are typical examples. To understand the structural basis and functional role of the native metastability of inhibitory serpins, we characterized stabilizing mutations of ␣ 1 -antitrypsin in a region presumably involved in complex formation with a target protease. We found various unfavorable interactions such as overpacking of side chains, polar-nonpolar interactions, and cavities as the structural basis of the native metastability. For several stabilizing mutations, there was a concomitant decrease in the inhibitory activity. Remarkably, some substitutions at Lys-335 increased the stability over 6 kcal mol ؊1 with simultaneous loss of activity over 30% toward porcine pancreatic elastase. Considering the location and energetic cost of Lys-335, we propose that this lysine plays a pivotal role in conformational switch during complex formation. Our current results are quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal mol ؊1 without any significant loss of activity. It appears that the local strain of inhibitory serpins is critical for the inhibitory activity.Facile conversion of the metastable native structure of proteins into an alternative more stable form, accompanying the execution of their functions, has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus (1), the strained native structure of plasma serpins (serine protease inhibitors) (2), and possibly the surface glycoprotein of human immunodeficiency virus (HIV) 1 (3) are typical examples. The native strain of serpins is considered to be crucial to their physiological functions, such as plasma protease inhibition (2, 4), hormone delivery (5), Alzheimer filament assembly (6, 7), and extracellular matrix remodeling (8). The inhibitory serpins, which include ␣ 1 -antitrypsin (␣ 1 AT), antithrombin III, ␣ 1 -antichymotrypsin, and plasminogen activator inhibitor-1, serve as a good model system to study the native metastability; several crystal structures of both the strained native (9 -13) and the relaxed cleaved forms (14 -16) are available. In addition, the inhibitory activity that presumably is related to the native metastability is easy to assay.The serpin structure is composed of three ␤-sheets and several ␣-helices (Fig. 1). Upon binding a target protease, the reactive center loop of inhibitory serpins is thought to be inserted into the major ␤-sheet, A sheet, to form a very stable complex between the inhibitor and the protease (17, 18). Various biochemical (19,20) and structural (21-23) studies suggest that the loop insertion is necessary for the formation of a stable complex but not sufficient to confer inhibitory activity. Instead, the rate of loop insertion is tho...

The role of human papilloma virus (HPV) infection in the development of cervical carcinoma is well established, however, the prevalence of HPV DNA in cervical adenocarcinoma varies from study to study. It appears to be caused by a number of factors, one of which is that cervical adenocarcinomas comprise a heterogeneous group of multiple subtypes. To clarify the impact of HPV infection on the development of cervical adenocarcinoma with diverse histological subtypes, we performed a population-based study in Korean women from 15 different institutes for the status of HPV infection in adenocarcinoma of uterine cervix. A total of 432 cervical adenocarcinomas from 1997 to 2001 were reviewed and classified according to the modified WHO classification. For 135 cases, HPV typing was performed with HPV DNA chip (82 cases) and PCR HPV typing (53 cases), using formalin-fixed, paraffin-embedded archival tissue. The overall prevalence of HPV infection in cervical adenocarcinoma was 90%. The infection of HPV 16 and/or HPV 18 accounted for 78% of HPV-positive adenocarcinomas. Multiple HPV types were found in 13% of the cases. The HPV DNA was rarely detected in minimal deviation adenocarcinoma. Interestingly, HPV 16 was a predominant type in endometrioid and villoglandular types, whereas HPV 16 and HPV 18 were detected with equal prevalence in other subtypes. In conclusion, HPV infection, mostly HPV 16 and HPV 18, is highly associated with most of the cervical adenocarcinomas, whereas endometrioid and villoglandular type have a different pattern of HPV infection status. Minimal deviation adenocarcinoma does not seem to be related with HPV infection. Modern Pathology (2005) 18, 528-534, advance online publication, 22 October 2004; doi:10.1038/modpathol.3800316Keywords: cervical adenocarcinoma; HPV infection; HPV DNA Chip; PCR-based HPV typingThe incidence and proportion of adenocarcinoma relative to squamous cell carcinoma in the uterine cervix has been increasing over the past several decades. Recent reports indicated that the adenocarcinoma accounted for 20-25% in uterine cervical cancer compared with only 5-15% in the past. [1][2][3] The epidemiologic risk for cervical adenocarcinoma is similar to those for invasive squamous cell carcinoma, such as multiple sexual partners and the early onset of sexual intercourse, which are related to the risk factors of human papilloma virus (HPV) infection. 4 Whereas the role of HPV infection in the development of cervical squamous cell carcinoma is well established, the pathogenetic role of the HPV in the cervical adenocarcinoma is still