Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis E virus.

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T=3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~440Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. & Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA108, 12681-12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.

Prevalence of nonpolypoid (flat and depressed) colorectal neoplasms in asymptomatic and symptomatic adults.

CONTEXT: Colorectal cancer is the second leading cause of cancer death in the United States. Prevention has focused on the detection and removal of polypoid neoplasms. Data are limited on the significance of nonpolypoid colorectal neoplasms (NP-CRNs). OBJECTIVES: To determine the prevalence of NP-CRNs in a veterans hospital population and to characterize their association with colorectal cancer. DESIGN, SETTING, AND PATIENTS: Cross-sectional study at a veterans hospital in California with 1819 patients undergoing elective colonoscopy from July 2003 to June 2004. MAIN OUTCOME MEASURES: Endoscopic appearance, location, size, histology, and depth of invasion of neoplasms. RESULTS: The overall prevalence of NP-CRNs was 9.35% (95% confidence interval [95% CI], 8.05%-10.78%; n = 170). The prevalence of NP-CRNs in the subpopulations for screening, surveillance, and symptoms was 5.84% (95% CI, 4.13%-8.00%; n = 36), 15.44% (95% CI, 12.76%-18.44%; n = 101), and 6.01% (95% CI, 4.17%-8.34%; n = 33), respectively. The overall prevalence of NP-CRNs with in situ or submucosal invasive carcinoma was 0.82% (95% CI, 0.46%-1.36%; n = 15); in the screening population, the prevalence was 0.32% (95% CI, 0.04%-1.17%; n = 2). Overall, NP-CRNs were more likely to contain carcinoma (odds ratio, 9.78; 95% CI, 3.93-24.4) than polypoid lesions, irrespective of the size. The positive size-adjusted association of NP-CRNs with in situ or submucosal invasive carcinoma was also observed in subpopulations for screening (odds ratio, 2.01; 95% CI, 0.27-15.3) and surveillance (odds ratio, 63.7; 95% CI, 9.41-431). The depressed type had the highest risk (33%). Nonpolypoid colorectal neoplasms containing carcinoma were smaller in diameter as compared with the polypoid ones (mean [SD] diameter, 15.9 [10.2] mm vs 19.2 [9.6] mm, respectively). The procedure times did not change appreciably as compared with historical controls. CONCLUSION: In this group of veteran patients, NP-CRNs were relatively common lesions diagnosed during routine colonoscopy and had a greater association with carcinoma compared with polypoid neoplasms, irrespective of size.

Studies on intracellular processing of the capsid protein of human astrovirus serotype 1 in infected cells.

Astroviruses are non-enveloped, positive-strand RNA viruses. Their structural (capsid) protein is processed extracellularly into several smaller fragments which are found on the mature viral particle. In addition, intracellular cleavage of the capsid protein has been proposed. However, analysis of capsid protein processing has been hampered by the lack of antibodies to regions near the N and C termini of the protein. Here we describe the construction of two infectious mutants of human astrovirus serotype 1 (HAstV-1), in which amino acids (aa) 11-30 or aa 783-787, respectively, of the 787 aa capsid protein were replaced by tag sequences. Processing of the tagged capsid proteins in infected Caco-2 cells was analysed by immunoprecipitation with specific reagents directed against the tags or against native internal regions of the capsid protein. No intracellular processing of the capsid protein in infected cells could be detected, while assembled viral particles were readily observed within cells.

Viral hepatitis and other infectious diseases in a homeless population.

GOALS: To determine the prevalence of four common infectious diseases-hepatitis B, hepatitis C, human immunodeficiency virus (HIV), and tuberculosis-as well as co-infection rates and risk factors in a homeless population. BACKGROUND: The prevalence of infectious diseases, especially viral hepatitis, among the homeless population is largely unknown. STUDY: This study consists of a retrospective analysis of the history and laboratory data collected from all homeless veterans admitted to a Veterans Administration (VA) domiciliary from May 1995 to March 2000. RESULTS: Of the homeless veterans admitted to a VA domiciliary program, 597 of 829 were screened for markers of all four infectious diseases. The overall prevalence of anti-hepatitis C virus (HCV) antibody, and positive result for purified protein derivative (PPD), anti-HIV antibody, and hepatitis B surface antigen (HbsAg) were 41.7%, 20.6%, 1.84% and 1.17%, respectively. At least one of the four markers was positive in 52.6% and more than one in 12%. Co-infection with HCV occurred commonly in veterans who were positive for anti-HIV (72.7%) and HBsAg (57.1%). Four self-reported major risk factors (intravenous drug use, alcohol abuse, previous imprisonment, and prior stay in a shelter) were evaluated. Multivariate analysis indicates that intravenous drug use and anti-HBs reactivity are independent risk factors for HCV infection, HCV infection for anti-hepatitis B surface antibody reactivity, and older age for PPD positivity. CONCLUSIONS: Chronic hepatitis C and co-infections are common among the homeless population. Patients infected with HIV and hepatitis B virus frequently are co-infected with HCV. Infections frequently are associated with certain identifiable risk factors.

Processing of nonstructural protein 1a of human astrovirus.

Astrovirus contains three open reading frames (ORF) on its genomic RNA, ORF1a, ORF1b, and ORF2. ORF1a encodes a 920-amino-acid (aa) nonstructural protein, nsP1a, which displays a 3C-like serine protease motif. Little is known about the processing of nsP1a or whether the protease it contains is active and involved in autocatalytic processing. Here we address both of these matters. Intact and N-terminally deleted forms of ORF1a from human astrovirus serotype 1 were expressed in BHK cells, and nsP1a-derived processing products were immunoprecipitated with an nsP1a-specific antibody or an antibody specific for an N-terminally linked epitope tag. The mapping of the main processing products, p20 and p27, suggests cleavage sites near aa 170, 410, and 655 of nsP1a. Cleavages at around aa 410 and 655, but not aa 170, were abolished when a 9-aa substitution was introduced into the protease motif in nsP1a. The p27 processing product was also found in Caco-2 cells that had been infected with human astrovirus serotype 1, confirming the presence of the cleavage sites at approximately aa 410 and 655.

Proteolytic processing of a human astrovirus nonstructural protein.

To analyse the activity of the putative 3C-like serine protease encoded in open reading frame (ORF)-1a of human astrovirus serotype 1 (HAstV-1), ORF-1a was transcribed and translated in vitro. Translation products, identified by immunoprecipitation with specific antibodies against recombinant C-terminal ORF-1a fragments, include the full-length 101 kDa (p101) protein and a 38 kDa band (p38). In addition, a 64 kDa protein (p64) was immunoprecipitated by an anti-FLAG antibody when a FLAG epitope was inserted at the N terminus of the ORF-1a product. Mutation of the amino acids predicted to form the catalytic triad of the HAstV-1 3C-like serine protease (Ser-551, Asp-489, His-461) resulted in undetectable levels of p38, supporting the involvement of the HAstV-1 3C-like serine protease and the importance of these amino acids in the processing of p101 into p38 and p64. N-terminal deletions of up to 420 aa of p101 that did not involve the predicted 3C-like serine protease motif did not alter levels of p38 compared to wild-type. C-terminal deletion analysis localized p38 to the C terminus of ORF-1a. Mutation of the P1 residue of the predicted cleavage site, which is conserved among known human and sheep astrovirus sequences, resulted in no detectable p38, supporting cleavage at the Gln-567/Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567/Thr-568 in a process that is dependent on the viral 3C-like serine protease.