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Fine-scale quantitative precipitation forecast is a key issue and challenge in weather forecasting services. In this study, based on hourly precipitation from the 1 km× 1 km resolution Southwest China WRF-based Intelligent Numeric Grid forecast System (SWC-WINGS), a fusion-corrected experiment was conducted using time lag and probability matching methods. The fusion-corrected forecast of hourly pre⁃ cipitation was then verified utilizing the CMA Multi-source Precipitation Analysis System (CMAPS) three-source merged precipitation obser⁃ vation grid data from 1 July to 31 August 2022 in Sichuan. Finally, the fusion-corrected method was applied to a short-term heavy precipita⁃ tion process over the western Sichuan Basin. The results show that: (1) Compared with the model precipitation forecasts, the time-lagged en⁃ semble forecast was over-optimistic for small-scale precipitation and over-conservative for large-scale precipitation. (2) However, the fu⁃ sion-corrected method by time lag and probability matching methods overcame the above difficulties and showed significant improvement in the TS score, particularly in 1~2 h nowcast time. The TS score for hourly precipitation exceeding 0.1 mm, 5 mm, 10 mm, and 20 mm were in⁃ creased on average by 7.2%, 17.2%, 28.3%, and 36.3%, respectively. (3) A case studies also showed that the fusion-corrected method had good improvement and correction capabilities on the hourly precipitation forecast, especially for large-scale precipitation forecasts.

Short-term precipitation forecasting is essential for agriculture, transportation, urban management, and tourism. The radar echo extrapolation method is widely used in precipitation forecasting. To address issues like forecast degradation, insufficient capture of spatiotemporal dependencies, and low accuracy in radar echo extrapolation, we propose a new model: MS-DD3D-RSTN. This model employs spatiotemporal convolutional blocks (STCBs) as spatiotemporal feature extractors and uses the spatial-temporal loss (STLoss) function to learn intra-frame and inter-frame changes for end-to-end training, thereby capturing the spatiotemporal dependencies in radar echo signals. Experiments on the Sichuan dataset and the HKO-7 dataset show that the proposed model outperforms advanced models in terms of CSI and POD evaluation metrics. For 2 h forecasts with 20 dBZ and 30 dBZ reflectivity thresholds, the CSI metrics reached 0.538, 0.386, 0.485, and 0.198, respectively, representing the best levels among existing methods. The experiments demonstrate that the MS-DD3D-RSTN model enhances the ability to capture spatiotemporal dependencies, mitigates forecast degradation, and further improves radar echo prediction performance.

The SARS-CoV-2 main protease (Mpro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature Mpro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 Mpro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released Mpro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released Mpro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 Mpro precursor autoprocessing or the released mature Mpro through different mechanisms of action.

Accurate wind speed prediction is significantly important for the full utilization of wind energy resources and the improvement in the economic benefits of wind farms. Because the ensemble forecast takes into account the uncertainty of information about the atmospheric motion, domestic and foreign weather service forecast centers often choose to use the ensemble numerical forecast to achieve the fine forecast of wind speed. However, due to the unavoidable systematic errors of the ensemble numerical forecast model, it is necessary to correct the deviation in the ensemble numerical forecast wind speed. Considering the typical spatio-temporal characteristics of the grid prediction data of the wind field, based on Convolutional Long–Short Term Memory (ConvLSTM) units and attention mechanism, this paper takes the complex and representative North China region as the research area, aiming to reveal the shortcomings of existing deep learning integrated prediction correction models in extracting temporal features of grid prediction data. We propose a new ensemble prediction wind field correction model integrating multi-factor and spatio-temporal characteristics. This model uses reanalyzed land data provided by the European Center for Medium-Range Weather Forecasts as the real data to correct the deviation in the near-surface 10 m wind field data predicted by the regional ensemble numerical prediction model of the China Meteorological Administration. We used the reanalyzed land data provided by the European Centre for Medium-Range Weather Forecasts (ECMWF) as the live data to correct the deviation in the near-surface 10 m wind field data predicted by the regional ensemble numerical forecast model of the China Meteorological Administration (CMA). At the same time, Root Mean Square Error (RMSE) and Mean Absolute Error (MAE) were used as the scoring indicators, and the results of the China Meteorological Administration–Regional Ensemble Prediction System (CMA–REPS) ensemble average, multiple linear regression method correction, Long–Short Term Memory (LSTM) method correction, and U-net (UNET) method correction were compared. Compared with the UNET model method, the experimental results show that when processing the 10 m zonal wind data, 10 m meridional wind data, and 10 m average wind speed data of CMA–REPS 24 h forecasts, the correction results of our model can reduce the RMSE score index by 9.15%, 4.83%, and 7.79%. At the same time, when processing the 48 h and 72 h near-surface 10 m wind field data of the CMA–REPS forecast, our model can improve the prediction accuracy of CMA–REPS near-surface wind forecast data. Therefore, the correction effect of the proposed model in a complex terrain area is evidently better compared to other methods.

Base on the precipitation forecast of ECWMF model from 2016 to 2019 and the observation data, five schemes are designed to carry out revision experiment on numerical model clear-rainy forecast, including Optimal Percentile (OP), the Optimal Threat Score (OTS), the Probability Matching (PM), the sub-district OTS and the sub-district PM-OTS fusion scheme. The results show that: (1) The clear-rainy revised thresholds of OP and PM are static, and that of OTS is dynamic. The thresholds of the 5 schemes are all applicable to Area A(basin, northern Aba Prefecture and northern Garze Prefecture), among which the thresholds of PM and sub-district PM-OTS are more applicable to Area B(southern Ganzi Prefecture and panxi area) with obvious wet deviation of the numerical model. (2) Each scheme has obvious revised ability for ECWMF model clear-rainy forecast. The revised effect of 24 h is the best, and that of Area B is superior to Area B, autumn and winter is better than spring and summer. (3) Revised scheme scores after subdivision are better than before, and the sub-district PM-OTS is the best. Individual cases and batch tests show that the revised effects of the schemes in Area A are comparable, the rainfall distribution in Area B are more close to the observed precipitation after revised of PM, sub-district OTS and PM-OTS fusion schemes, among which the sub-district PM-OTS is the best.

Capable of retaining rainwater, Organic Ground Cover (OGC) is an ecological material for urban greening. The water absorption features of various OGC materials should be identified before selecting the proper OGC for places under different climates. With the aid of a rainfall simulator, this paper experimentally explores the water absorption features of 16 common OGC materials at the rainfall intensity of 48 mm/h. Based on the experimental results, the change trends of multiple indices were analyzed for these materials, namely, volumetric water absorption ratio, volumetric water absorption rate, mass water absorption ratio, mass water absorption rate, pre-drainage duration, and near saturation duration. The results show that the OGC materials differed significantly in water absorption capacity: (1) SD and PPB2 had the best volumetric water absorption ratios, while EC had the worst volumetric water absorption ratio. (2) The volumetric water absorption rates of all materials reached the maximum in 0–10 min after the artificial rainfall, and then decreased rapidly; At 10 min, the proportion of volumetric water absorption ratios of the 16 materials averaged at 74%; The highest (94%) and lowest (36%) proportions belong to OC and SD, respectively. (3) OC had the best mass water absorption ratio, while EC had the worst mass water absorption ratio. (4) At 10 min, the proportion of mass water absorption ratio of each material was the same as that of volumetric water absorption ratio; OC, PN, CF, CFP, PPB1, WB, and ANBM had obviously higher mass water absorption rates than other materials, while PS, FPB2 and EC had relatively small mass water absorption rates. (5) The shortest and longest pre-drainage durations belong to OC (2 min) and SD (13.67 min), respectively. (6) The shortest pre-near saturation belongs to CFP, PPB1, FPB2 and EC (13.33 min), the longest pre-near saturation belongs to SD (50 min), respectively. The research results provide a reference for the selection of the OGC materials in urban greening.

In urban green spaces, it is important to maintain a suitable soil moisture content, an important indicator of plant growth. The management of urban green spaces faces great challenges in semi-humid areas of China, which are featured by interannual changes in rainfall and the alternation between droughts and floods. This paper aims to select suitable Organic Ground Cover (OGC) materials for urban green spaces in such areas, as the OGC can effectively absorb water and suppress the evaporation of soil moisture. For this purpose, 16 OGC materials were selected for our experiments, and 33 identical holes were dug on a field of silty loam. Next, 16 holes were separately covered by a 5 cm-thick layer of the 16 materials, 16 holes were separately covered by a 10 cm-thick layer of the 16 materials, and 1 hole was taken as the control sample (CK). On this basis, the authors explored how the 16 materials in two different thicknesses affect the soil moisture contents in four different soil depths. The results show that: When the OGC layer was 5 cm thick and 10 cm thick, CK had the lowest soil moisture content at any soil depth. In most cases, the soil moisture content under the same OGC material changed insignificantly with the material thicknesses, except FPB2 and CP. The layer of OGC materials should be 5 cm, if the only purpose is to increase the soil moisture content and enhance the landscape effect, and 10 cm if rainwater absorption is also taken into account. The above results provide a reference for selecting the OGC materials suitable for urban greening.

HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.

HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

Abstract In this paper, some recursion formulae of sums for the Riemann Zeta function and Dirchlet, series are obtained through expanding several simple function on [-π,π] or [0, 2π] by [...]

Abstract We present some inequalities for the polygamma funtions. As an application, we give the upper and lower bounds for the expression [n.summation over (k=1)] 1/k - 1n n - [...]

The aim of this paper is to establish some asymptotic expansions and inequalities related to BBP- and Ramanujan-type approximation series of the constant. [ABSTRACT FROM AUTHOR]

Abstract Background Rice is a typical monocotyledonous plant and an important cereal crop. The structural units of rice flowers are spikelets and florets, and floral organ development and spike germination affect rice reproduction and yield. Results In this study, we identified a novel long sterile lemma (lsl2) mutant from an EMS population. First, we mapped the lsl2 gene between the markers Indel7–22 and Indel7–27, which encompasses a 25-kb region. The rice genome annotation indicated the presence of four candidate genes in this region. Through gene prediction and cDNA sequencing, we confirmed that the target gene in the lsl2 mutant is allelic to LONG STERILE LEMMA1 (G1)/ELONGATED EMPTY GLUME (ELE), hereafter referred to as lsl2. Further analysis of the lsl2 and LSL2 proteins showed a one-amino-acid change, namely, the mutation of serine (Ser) 79 to proline (Pro) in lsl2 compared with LSL2, and this mutation might change the function of the protein. Knockout experiments showed that the lsl2 gene is responsible for the long sterile lemma phenotype. The lsl2 gene might reduce the damage induced by spike germination by decreasing the seed germination rate, but other agronomic traits of rice were not changed in the lsl2 mutant. Taken together, our results demonstrate that the lsl2 gene will have specific application prospects in future rice breeding. Conclusions The lsl2 gene is responsible for the long sterile lemma phenotype and might reduce the damage induced by spike germination by decreasing the seed germination rate.

Background: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. Results: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. Conclusions: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor. [ABSTRACT FROM AUTHOR]

In order to identify objective parameters for quality evaluation, physical dimensions, pH, color parameters, proximate composition, water activity, lipid oxidation, free amino acids, mechanical properties, protein composition and differential scanning calorimetry of commercial Japanese spicy pollack roe products of differing quality were analyzed. Low-priced roe products showed significantly higher (p < 0.05) average thiobarbituric acid reactive substances content than high-priced roe products from the same company. More bitter amino acids were detected in the low-priced, compared to the high-priced roe products from the same company. Ovary membrane and eggshell proteins in low-priced roe products were comprised of a relatively greater amount of low-molecular-weight components. Compared to low-priced roe products, high-priced ones showed higher values for the mechanical properties and thermal transition enthalpy of fish egg. [ABSTRACT FROM AUTHOR]

Background HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. Results Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. Conclusions The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways. [ABSTRACT FROM AUTHOR]

Let γ = 0.577215 ... be the Euler-Mascheroni constant, and let Pn = Σ kn = 1 1/k - 1/2 ln (n2 + n + 1/3) and Qn = Σ kn = 1 1/k - 1/4 ln [ (n2+n2+1/3)2-1/45] We prove that for all integers n ⩾ 1, 1/180(n+1)4 < γ - Pn < 1/180n4 and 8/2835(n+1)6 < Qn-γ < 8/2835n6. This provides the higher order estimate for the Euler-Mascheroni constant. [ABSTRACT FROM AUTHOR]

Background: HIV protease (PR) is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results: We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR), and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions: We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing. [ABSTRACT FROM AUTHOR]

We present the monotonicity and logarithmic convexity properties of the functions involving the gamma function. Minc-Sathre inequality are extended and refined. [ABSTRACT FROM AUTHOR]

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the μ2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an ∼300-residue region of AIP1/Alix-(409–715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 μ2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant μ2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAPL domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities. [ABSTRACT FROM AUTHOR]

Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymeriziug drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production. [ABSTRACT FROM AUTHOR]

Attempts to define specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus. Use of full-length provirus replication and Gag/Gag-Pol expression systems; Evidence that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-paired GC stretch, and a pseudoknot structure.

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAVUK viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain. [ABSTRACT FROM AUTHOR]

Retroviral Gag polyproteins are necessary and sufficient for virus budding. Numerous studies of human immunodeficiency virus type 1 (HIV-1) Gag assembly and budding mechanisms have been reported, but relatively little is known about these fundamental pathways among animal lentiviruses. While there may be a general assumption that lentiviruses share common assembly mechanisms, studies of equine infectious anemia virus (EIAV) have indicated alternative cellular pathways and cofactors employed among lentiviruses for assembly and budding. In the current study, we used bimolecular fluorescence complementation to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells. The results of these studies demonstrated that replacing the natural RNA nuclear export element (Rev-response element [RRE]) used by HIV-1 and EIAV with the hepatitis B virus posttranscriptional regulatory element (PRE) altered HIV-1, but not EIAV, Gag assembly sites and budding efficiency in human cells. Consistent with this novel observation, different assembly sites were revealed in human cells for Rev-dependent EIAV and HIV-1 Gag polyproteins. In rodent cells, Rev-dependent HIV-1 Gag assembly and budding were blocked, but changing RRE to PRE rescued HIV-1 Gag assembly and budding. In contrast, EIAV Gag polyproteins synthesized from mRNA exported via either Rev-dependent or PRE-dependent mechanisms were able to assemble and bud efficiently in rodent cells. Taken together, our results suggest that lentivirus assembly and budding are regulated by the RNA nuclear export pathway and that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding. [ABSTRACT FROM AUTHOR]

Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S-transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli. Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PRH69E) folds into active enzymes, and it does so with an apparent Kd (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PRH69E and suggesting a folding defect in the precursor. [ABSTRACT FROM AUTHOR]

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-1ike domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain. [ABSTRACT FROM AUTHOR]

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPXnL L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPXnL-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif. [ABSTRACT FROM AUTHOR]