Citations

ABSTRACT Productive replication of human immunodeficiency virus type 1 (HIV-1) occurs efficiently only in humans. The posttranscriptional stages of the HIV-1 life cycle proceed poorly in mouse cells, with a resulting defect in viral assembly and release. Previous work has shown that the presence of human chromosome 2 increases HIV-1 production in mouse cells. Recent studies have shown that human chromosome region maintenance 1 (hCRM1) stimulates Gag release from rodent cells. Here we report that expressions of hCRM1 in murine cells resulted in marked increases in the production of infectious HIV-1 and feline immunodeficiency virus (FIV). HIV-1 production was also increased by hSRp40, and a combination of hCRM1 and hSRp40 resulted in a more-than-additive effect on HIV-1 release. In contrast, the overexpression of mouse CRM1 (mCRM1) minimally affected HIV-1 and FIV production and did not antagonize hCRM1. In the presence of hCRM1 there were large increases in the amounts of released capsid, which paralleled the increases in the infectious titers. Consistent with this finding, the ratios of unspliced to spliced HIV-1 mRNAs in mouse cells expressing hCRM1 and SRp40 became similar to those of human cells. Furthermore, imaging of intron-containing FIV RNA showed that hCRM1 increased RNA export to the cytoplasm.By testing chimeras between mCRM1 and hCRM1 and comparing those sequences to feline CRM1, we mapped the functional domain to HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats 4A to 9A and a triple point mutant in repeat 9A, which showed a loss of function. Structural analysis suggested that this region of hCRM1 may serve as a binding site for viral or cellular factors to facilitate lentiviral RNA nuclear export.

Abstract Microarray gene expression profiling has facilitated identification of molecular subtypes which can be associated with disease outcome and treatment response. However current platforms lack disease focus, potentially missing vital information contained in patient tissue samples. Moreover, a wide range of FFPE sample cohorts are available but their use is limited by the mRNA degradation inherent to the sample fixation process. By combining the Breast Cancer DSA® with powerful data analysis methodology, we reproduced the five major molecular subtypes in breast cancer based on a cohort of 107 FFPE samples. Furthermore, we showed that the established subtypes do not extend to BRCA1 and BRCA2 mutant tumours, necessitating expansion of the original breast cancer subtypes definition. Following RMA pre-processing of the 107 raw profiles, the data was transformed to overcome the association between profile quality and expression variability. If left untreated, this technical variability, often observed with FFPE tissues, can impair the study of inherent biological variations. Using the intrinsic list of 277 genes previously identified as differentiating breast cancer subtypes in fresh frozen samples [Sorlie et al., 2003], initially the sporadic samples were examined using hierarchical agglomerative clustering. All five original subtypes were identified, verifying the ability to detect subtypes in FFPE samples using the breast cancer DSA®. When the intrinsic list was applied to all 107 samples including BRCA1 and BRCA2 mutant samples, the subtypes were still distinguishable, although 31% of the mutant tumors were unclassified suggesting that specific biological processes defining BRCA1/BRCA2 mutant tumours are not captured by the original breast cancer molecular classification. We have successfully overcome the limitation imposed by FFPE sample microarray profiling by using the Breast Cancer DSA® and advanced data manipulation and exploration to reproduce molecular subtypes previously identified using fresh frozen samples. Further investigation to extend the previously identifed subtypes will progress the investigation of the heterogeneous biology underlying breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2011-326

ABSTRACT Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.

We tested the hypothesis that diaphragm muscle shortening modulates volume displacement and kinematics of the lower rib cage in dogs and that posture and mode of ventilation affect such modulation. Radiopaque markers were surgically attached to the lower three ribs of the rib cage and to the midcostal region of the diaphragm in six dogs of ∼8 kg body masses, and the locations of these markers were determined by a biplane fluoroscopy system. Three-dimensional software modeling techniques were used to compute volume displacement and surface area of the midcostal diaphragm and the lower three ribs during quiet spontaneous breathing, mechanical ventilation, and bilateral phrenic nerve stimulation at different lung volumes spanning the vital capacity. Volume displaced by the diaphragm relative to that displaced by the lower ribs is disproportionately greater under mechanical ventilation than during spontaneous breathing in the supine position ( P < 0.05). At maximal stimulation, diaphragm volume displacement grows disproportionately larger than rib volume displacement as lung volume increases ( P < 0.05). Surface area of both the diaphragm and the lower ribs during maximal stimulation of the diaphragm is reduced compared with that at spontaneous breathing ( P < 0.05). In the prone posture, mechanical ventilation results in a smaller change in diaphragm surface area than spontaneous breathing ( P < 0.05). Our data demonstrate that during inspiration the lower rib cage moves not only through the pump- and bucket-handle motion, but also rotates around the spine. Taken together, these data support the observation that the kinematics of the lower rib cage and its mechanical interaction with the diaphragm are more complex than previously known.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag and genomic RNA determinants required for encapsidation are well established, but where and when encapsidation occurs in the cell is unknown. We constructed MS2 phage coat protein labeling systems to track spatial dynamics of primate and nonprimate lentiviral genomic RNAs (HIV-1 and feline immunodeficiency virus [FIV]) vis-à-vis their Gag proteins in live cells. Genomic RNAs of both lentiviral genera were observed to traffic into the cytoplasm, and this was Rev dependent. In transit, FIV Gag and genomic RNA accumulated independently of each other at the nuclear envelope, and focal colocalizations of genomic RNA with an intact packaging signal (ψ) and Gag were observed to extend outward from the cytoplasmic face. In contrast, although HIV-1 genomic RNA was detected at the nuclear envelope, HIV-1 Gag was not. For both lentiviruses, genomic RNAs were seen at the plasma membrane if and only if Gag was present and ψ was intact. In addition, HIV-1 and FIV genomes accumulated with Gag in late endosomal foci, again, only ψ dependently. Thus, lentiviral genomic RNAs require specific Gag binding to accumulate at the plasma membrane, packaged genomes cointernalize with Gag into the endosomal pathway, and plasma membrane RNA incorporation by Gag does not trigger committed lentiviral particle egress from the cell. Based on the FIV results, we hypothesize that the Gag-genome association may initiate at the nuclear envelope.

ABSTRACT Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis -acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans . When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5′ leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.