Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a high rate of mortality. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent of the disease. Although public health procedures have been effective in combating the spread of SARS, concern remains about the possibility of a recurrence. Various approaches are being pursued for the development of efficacious therapeutics. One promising approach is to develop small molecule inhibitors of the essential major polyprotein processing protease 3Clpro. Here we report a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160,000 possible naturally occurring tetrapeptides. The substrate specificity data show the expected P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln. These data were then used to develop optimal substrates for a high-throughput screen of a 2000 compound small-molecule inhibitor library consisting of known cysteine protease inhibitor scaffolds. We also report the 1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor. This inhibitor, an alpha,beta-epoxyketone, inhibits 3Clpro with a k3/Ki of 0.002 microM(-1) s(-1) in a mode consistent with the substrate specificity data. Finally, we report the successful rational improvement of this scaffold with second generation inhibitors. These data provide the foundation for a rational small-molecule inhibitor design effort based upon the inhibitor scaffold identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region.

OBJECTIVELateral lumbar interbody fusion (LLIF) is a less invasive surgical option commonly used for a variety of spinal conditions, including in high-risk patient populations. LLIF is often performed as a stand-alone procedure, and may be complicated by graft subsidence, the clinical ramifications of which remain unclear. The aim of this study was to characterize further the sequelae of graft subsidence following stand-alone LLIF.METHODSA retrospective review of prospectively collected data was conducted on consecutive patients who underwent stand-alone LLIF between July 2008 and June 2015; 297 patients (623 levels) met inclusion criteria. Imaging studies were examined to grade graft subsidence according to Marchi criteria, and compared between those who required revision surgery and those who did not. Additional variables recorded included levels fused, DEXA (dual-energy x-ray absorptiometry) T-score, body mass index, and routine demographic information. The data were analyzed using the Student t-test, chi-square analysis, and logistic regression analysis to identify potential confounding factors.RESULTSOf 297 patients, 34 (11.4%) had radiographic evidence of subsidence and 18 (6.1%) required revision surgery. The median subsidence grade for patients requiring revision surgery was 2.5, compared with 1 for those who did not. Chi-square analysis revealed a significantly higher incidence of revision surgery in patients with high-grade subsidence compared with those with low-grade subsidence. Seven of 18 patients (38.9%) requiring revision surgery suffered a vertebral body fracture. High-grade subsidence was a significant predictor of the need for revision surgery (p < 0.05; OR 12, 95% CI 1.29–13.6), whereas age, body mass index, T-score, and number of levels fused were not. This relationship remained significant despite adjustment for the other variables (OR 14.4; 95% CI 1.30–15.9).CONCLUSIONSIn this series, more than half of the patients who developed graft subsidence following stand-alone LLIF required revision surgery. When evaluating patients for LLIF, supplemental instrumentation should be considered during the index surgery in patients with a significant risk of graft subsidence.