The Glycosylated Gag Protein of a Murine Leukemia Virus Inhibits the Antiretroviral Function of APOBEC3

APOBEC proteins have evolved as innate defenses against retroviral infections. Human immunodeficiency virus (HIV) encodes the Vif protein to evade human APOBEC3G; however, mouse retroviruses do not encode a Vif homologue, and it has not been understood how they evade mouse APOBEC3. We report here a murine leukemia virus (MuLV) that utilizes its glycosylated Gag protein (gGag) to evade APOBEC3. gGag is critical for infection of in vitro cell lines in the presence of APOBEC3. Furthermore, a gGag-deficient virus restricted for replication in wild-type mice replicates efficiently in APOBEC3 knockout mice, implying a novel role of gGag in circumventing the action of APOBEC3 in vivo.APOBEC3G (hA3G) in humans and its mouse orthologue, APOBEC3 (mA3), act as potent innate defenses against retroviral infection. Both proteins deaminate cytidine in single-stranded DNA, ultimately resulting in hypermutation of newly synthesized proviral DNA (6, 16), although additional deaminase-independent mechanisms of inhibition have been identified (2). Infectious exogenous retroviruses, including human immunodeficiency virus (HIV) and murine leukemia viruses (MuLVs), have evolved mechanisms to circumvent the action of the APOBEC proteins (3, 6). HIV encodes the Vif protein, which facilitates the rapid proteolysis of hA3G, while the mechanism by which exogenous MuLVs evade the action of mA3 is unknown (6).Exogenous MuLVs, as well as some other gammaretroviruses, encode a glycosylated Gag protein (gGag) originating from an alternate translation start site upstream of the methionine start site of the Gag structural polyproteins (10, 17, 27). gGag is synthesized at similar rates and levels as the structural Gag polyprotein in MuLV-infected cells but is glycosylated and undergoes distinct proteolytic processing (10, 12, 21). A carboxyl fragment of gGag is released from the cell, while an amino fragment is incorporated into the plasma membrane as a type 2 transmembrane protein (12, 25). The functions of gGag remain unclear, but mutations that eliminate its synthesis severely impede in vivo replication of the virus with little, if any, effect on replication in fibroblastic cell lines (7, 19, 26). APOBEC3 proteins are expressed in many tissues in vivo but are poorly expressed in many in vitro cell lines (6), suggesting a possible link between gGag expression and the evasion of mA3 by MuLVs. These studies were undertaken to determine if the expression of the gGag protein facilitated MuLV replication in the presence of mA3 in vitro and in vivo.     

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne''s disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne''s disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.     

Cross-Ecosystem Comparisons of In Situ Plant Uptake of Amino Acid-N and NH4 +

Plant and microbial use of nitrogen (N) can be simultaneously mutualistic and competitive, particularly in ecosystems dominated by mycorrhizal fungi. Our goal was to quantify plant uptake of organic and inorganic N across a broad latitudinal gradient of forest ecosystems that varied with respect to overstory taxon, edaphic characteristics, and dominant mycorrhizal association. Using ¹³C and ¹⁵N, we observed in situ the cycling dynamics of NH₄ ⁺ and glycine through various soil pools and fine roots over 14 days. Recovery of ¹⁵N as soil N varied with respect to N form, forest type, and sampling period; however, there were similarities in the cycling dynamics of glycine and NH₄ ⁺ among all forest types. Microbial immobilization of ¹⁵N was immediately apparent for both treatments and represented the largest sink (~25%) for ¹⁵N among extractable soil N pools during the first 24 h. In contrast, fine roots were a relatively small sink (<10%) for both N forms, but fine root ¹³C enrichment indicated that plants in all forest types absorbed glycine intact, suggesting that plants and microbes effectively target the same labile soil N pools. Relative uptake of amino acid-N versus NH₄ ⁺ varied significantly among sites and approximately half of this variation was explained by mycorrhizal association. Estimates of plant uptake of amino acid-N relative to NH₄ ⁺ were 3× higher in ectomycorrhizal-dominated stands (1.6 ± 0.2) than arbuscular mycorrhizae-dominated stands (0.5 ± 0.1). We conclude that free amino acids are an important component of the N economy in all stands studied; however, in these natural environments plant uptake of organic N relative to inorganic N is explained as much by mycorrhizal association as by the availability of N forms per se.     

Profound amplification of pathogenic murine polytropic retrovirus release from coinfected cells

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.     

Fibrotic lesions in the testis of bulls and relationship to semen quality

Ultrasonography of the testes was done in bulls at three locations in western Canada (n = 325) and one in Argentina (n = 387) to determine the prevalence of fibrotic lesions and to examine the relationship between fibrotic lesions and location, age, breed, right compared with left testes, testis size and semen quality. Fibrotic lesions were common in the testes of bulls raised under intensive rearing conditions in western Canada as well as in the more extensive rearing conditions of Argentina. Fibrotic lesions appeared as early as 5–6 months of age and the number of cases continued to increase until at least 12–14 months of age. The severity of lesions increased in some cases during this period; however, it appears that the development of lesions occurred during a finite period of pubertal development. It is unlikely that the prevalence of lesions is influenced by breed, right compared with left testes or testis size. The cause of the lesions is unknown, but there was an association between the development of fibrotic lesions and an outbreak of BRSV disease in Argentina in one group of bulls. There was some indication that during the active process that leads to fibrosis spermatogenesis is adversely affected; however, the presence of a large number of fibrotic lesions that may occupy as much as 50% of the testis parenchyma did not preclude the production of a greater percentage of sperm with normal morphology.     

Parallel synthesis of a series of potentially brain penetrant aminoalkyl benzoimidazoles

Alpha7 agonists were identified via GOLD (CCDC) docking in the putative agonist binding site of an alpha7 homology model and a series of aminoalkyl benzoimidazoles was synthesised to obtain potentially brain penetrant drugs. The array was prepared starting from the reaction of ortho-fluoronitrobenzenes with a selection of diamines, followed by reduction of the nitro group to obtain a series of monoalkylated phenylene diamines. N,N'-Carbonyldiimidazole (CDI) mediated acylation, followed by a parallel automated work-up procedure, afforded the monoacylated phenylenediamines which were cyclised under acidic conditions. Parallel work-up and purification afforded the array products in good yields and purities with a robust parallel methodology which will be useful for other libraries. Screening for alpha7 activity revealed compounds with agonist activity for the receptor.     

Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis

Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 °C to 32 °C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 °C, at 32 °C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sensory evaluation of character impact components in an apple model mixture

Food aromas generally are complex mixtures of volatiles. In the present study, we investigated the joint effects of hexyl acetate, trans-2-hexenal and 1-hexanol on the multi-attribute perception of an apple aroma. The first two substances were identified earlier as positive contributors to the apple aroma (high character impact), whereas the third component was identified as an irrelevant or negative contributor (low character impact). Aroma quality was quantified using a set of eight graphic rating scales. All three components had significant effects on the aroma profiles. These effects consist mainly of an effect of each component on the attribute that described its individual character and an effect of all three components on ratings on the main character attribute 'apple'. As expected, the high impact components increased 'apple' ratings, whereas the low character impact component decreased 'apple' ratings. Furthermore, intensity ratings on the attribute that corresponded with the odour of the low impact component were suppressed by the presence of high impact components. These results indicate that the contributions of odorants to the mixture's aroma are not linear combinations of separate odour intensities, because sensory interactions were observed. In addition, humans detect components in complex mixtures more accurately than studies on identification performance have suggested. We conclude that for an adequate assessment of the effects of multiple mixture components on changes in aroma perception, it is sufficient to employ multiple response scales measuring intensities of attributes that are distinctive with respect to the expected qualitative changes. Results of this approach should be subjected to multivariate methods of statistical analysis.     

Hemlock Declines Rapidly with Hemlock Woolly Adelgid Infestation: Impacts on the Carbon Cycle of Southern Appalachian Forests

The recent infestation of southern Appalachian eastern hemlock stands by hemlock woolly adelgid (HWA) is expected to have dramatic and lasting effects on forest structure and function. We studied the short-term changes to the carbon cycle in a mixed stand of hemlock and hardwoods, where hemlock was declining due to either girdling or HWA infestation. We expected that hemlock would decline more rapidly from girdling than from HWA infestation. Unexpectedly, in response to both girdling and HWA infestation, hemlock basal area increment (BAI) reduced substantially compared to reference hardwoods in 3 years. This decline was concurrent with moderate increases in the BAI of co-occurring hardwoods. Although the girdling treatment resulted in an initial pulse of hemlock needle inputs, cumulative litter inputs and O horizon mass did not differ between treatments over the study period. Following girdling and HWA infestation, very fine root biomass declined by 20–40% in 2 years, which suggests hemlock root mortality in the girdling treatment, and a reduction in hemlock root production in the HWA treatment. Soil CO₂ efflux (E _soil) declined by approximately 20% in 1 year after both girdling and HWA infestation, even after accounting for the intra-annual variability of soil temperature and moisture. The reduction in E _soil and the concurrent declines in BAI and standing very fine root biomass suggest rapid declines in hemlock productivity from HWA infestation. The accelerated inputs of detritus resulting from hemlock mortality are likely to influence carbon and nutrient fluxes, and dictate future patterns of species regeneration in these forest ecosystems. AEN performed research and analyzed data; NW performed research, analyzed data, and wrote the article; CRF contributed new methods, analyzed data, and wrote the article; RLH designed the study; JMV conceived of and designed the study; and BDK performed research.