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PubMed1997

Melanie II--a third-generation software package for analysis of two-dimensional electrophoresis images: II. Algorithms.

After two generations of software systems for the analysis of two-dimensional electrophoresis (2-DE) images, a third generation of such software packages has recently emerged that combines state-of-the-art graphical user interfaces with comprehensive spot data analysis capabilities. A key characteristic common to most of these software packages is that many of their tools are implementations of algorithms that resulted from research areas such as image processing, vision, artificial intelligence or machine learning. This article presents the main algorithms implemented in the Melanie II 2-D PAGE software package. The applications of these algorithms, embodied as the feature of the program, are explained in an accompanying article (R. D. Appel et al.; Electrophoresis 1997, 18, 2724-2734).

Algorithms↗

PubMed2001

A comparison of software packages that assess linkage using a variance components approach.

We report single-point and multipoint results from three statistical genetics software packages using a variance components approach for quantitative traits. When possible, we have shown the effects of covariates. Results from GENEHUNTER, SOLAR, and ACT software packages are compared for the quantitative trait immunoglobulin E (IgE) using chromosome 5 asthma familial data from Oxfordshire (England), Perth (Australia), and Freiburg (Germany), and using the genome-wide German data. Although few differences among variance components results for GENEHUNTER, SOLAR, and ACT were detected on a limited region of chromosome 5, these packages showed larger disagreement in the German data for chromosomes 1, 9, and 14.

Adult↗

PubMed1999

Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis.

Dominant negative (DN) mutants of the hepadnaviral core protein are potent inhibitors of viral replication. We have previously shown that fusion of sequences derived from the duck hepatitis B virus (DHBV) polymerase (Pol), DHBV small surface protein (S), bacterial beta-galactosidase (lacZ), or green fluorescent protein (GFP) to the carboxy terminus of the DHBV core protein yields DN mutants that inhibit viral replication at the posttranslational level. To elucidate the mechanism(s) of their antiviral action, we analyzed the effect of the DN mutants on RNA pregenome packaging and nucleocapsid assembly. Core-Pol and core-S, but not core-lacZ or core-GFP, markedly interfered with RNA pregenome packaging. Nucleocapsid formation was not affected by any of the mutants. The DN core-GFP fusion protein formed mixed particles with wild-type core protein in the cytoplasm of cotransfected cells and interfered with reverse transcription of the viral pregenome. A subpopulation of chimeric nucleocapsids, however, was shown to overcome the block in DNA synthesis and produce mature viral DNA. Thus, at least 2 steps within the viral life cycle can be targeted by DN DHBV core proteins: 1) packaging of the viral pregenome; and 2) reverse transcription within mixed particles. The fact that some mixed particles retain replication competence demonstrates a high structural flexibility of nucleocapsids and indicates a possible mechanism of viral escape.

Animals↗

PubMed1987

Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.

Human adenovirus type 5 (Ad5) contains a 36-kb double-stranded DNA molecule in an icosahedral capsid. Attempts to construct Ad5 insertion mutants containing DNA of more than about 105% of the genome size resulted in viral progeny in which deletions had occurred suggesting the existence of severe constraints on the size of packageable DNA molecules. To partially circumvent these constraints we used an adenovirus vector, Ad5dlE1,3, with deletions in early regions 1 (E1) and 3 for a total net reduction in genome size of 5349 bp and an expected capacity for inserts of greater than 7 kb. To use this vector efficiently we generated a circular form of dlE1,3 DNA which could be propagated as an infectious bacterial plasmid. When this plasmid was used as a recipient for inserts of various sizes it was found that its capacity was much less than expected and that dlE1,3 virion capsids could not even package DNA as large as the wt genome. Because the E1 deletion of dlE1,3 extends into the coding sequences for protein IX, a minor capsid component known to affect the heat stability of adenovirions, the possibility that absence of this polypeptide might also affect the DNA capacity of the virion was investigated. It was found that when the coding sequences for protein IX were restored the packaging capacity of the vector was also restored to that of wt virions. Thus protein IX is an essential constituent of virion capsids dispensable only for virions containing DNA of less than genomic size.

Adenovirus Early Proteins↗

PubMed1990

A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA.

The selective encapsidation of the hepadnaviral RNA pregenome from an excess of other viral and cellular mRNAs predicts specific protein-RNA interactions involving one or several sites on the pregenome. Using deletion analysis in a transient expression/packaging system in which all relevant viral proteins are provided in trans from a packaging-deficient helper genome, we identified near the 5'-end of the pregenome a 137 nucleotide sequence that is necessary and sufficient for RNA encapsidation; other parts of the 3 kb pregenome were found not to contribute to this process. The encapsidation sequence, which we call epsilon, possesses several interesting features with implications for the pregenome's function in RNA packaging, RNA translation and reverse transcription. (i) epsilon contains several indirect repeat sequences, suggesting a high degree of secondary structure, (ii) epsilon overlaps with the start signal for core gene translation, suggesting a mechanism to regulate the alternative use of the pregenome as core mRNA, (iii) epsilon does not contain the direct repeat sequences known to be involved in the initiation of viral DNA synthesis. Finally, our deletion analysis suggests that ribosomes translating the epsilon sequence from the precore start codon may interfere with genomic RNA packaging.

Base Sequence↗

PubMed1992

Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.

The Rous sarcoma virus (RSV) RNA leader sequence carries three open reading frames (uORFs) upstream of the AUG initiator of the gag gene. We studied, in vivo, the role of these uORFs by changing two or three nucleotides of the three AUGs or by deleting the first uORF. Our results show that (i) unlike most previously characterized uORFs, which decrease translation, the first uORF (AUG1) of RSV acts as an enhancer of translation, since absence of the first AUG decreased translation; AUG3 also modulates translation, probably by interfering with scanning ribosomes as described for other upstream ORFs, and mutation of AUG2 had no effect on translation. (ii) Mutation of each of the upstream AUGs lowered the infectivity of progeny virions. (iii) Unexpectedly, mutation of AUG1 and/or AUG3 dramatically reduced RNA packaging by 50-to 100-fold, unlike mutation of AUG2 which did not alter RNA packaging efficiency. Additional mutants in the vicinity of uORF1 and uORF3 were constructed in order to elucidate the mechanism by which uORFs affect RNA packaging: a translation model requiring uORFs 1 and 3, and involving ribosome pausing at AUG 3 is discussed.

Amino Acid Sequence↗

PubMed2005

Control of adenovirus packaging.

The results of studies of Adenovirus have contributed to our basic understanding of the molecular biology of the cell. While a great body of knowledge has been developed concerning Ad gene expression, viral replication, and effects on the infected host, the molecular details of the assembly process of Adenovirus particles are largely unknown. In this article, we would like to propose a theoretical model for the packaging and assembly of Adenovirus and present an overview of the studies that have contributed to our present understanding. In particular, we will summarize the molecular details of the process for packaging of viral DNA into virus particles and highlight the events in packaging and assembly that require further study.

Adenoviridae↗

PubMed2004

An overview of polylactides as packaging materials.

Polylactide polymers have gained enormous attention as a replacement for conventional synthetic packaging materials in the last decade. By being truly biodegradable, derived from renewable resources and by providing consumers with extra end-use benefits such as avoiding paying the "green tax" in Germany or meeting environmental regulations in Japan, polylactides (PLAs) are a growing alternative as a packaging material for demanding markets. The aim of this paper is to review the production techniques for PLAs, summarize the main properties of PLA and to delineate the main advantages and disadvantages of PLA as a polymeric packaging material. PLA films have better ultraviolet light barrier properties than low density polyethylene (LDPE), but they are slightly worse than those of cellophane, polystyrene (PS) and poly(ethylene terephthalate) (PET). PLA films have mechanical properties comparable to those of PET and better than those of PS. PLA also has lower melting and glass transition temperatures than PET and PS. The glass transition temperature of PLA changes with time. Humidity between 10 and 95% and storage temperatures of 5 to 40 degrees C do not have an effect on the transition temperature of PLA, which can be explained by its low water sorption values (i.e. <100 ppm at Aw = 1). PLA seals well at temperatures below the melting temperature but an appreciable shrinking of the films has been noted when the material is sealed near its melting temperature. Solubility parameter predictions indicate that PLA will interact with nitrogen compounds, anhydrides and some alcohols and that it will not interact with aromatic hydrocarbons, ketones, esters, sulfur compounds or water. The CO2, O2 and water permeability coefficients of PLA are lower than those of PS and higher than those of PET. Its barrier to ethyl acetate and D-limonene is comparable to PET. The amount of lactic acid and its derivatives that migrate to food simulant solutions from PLA is much lower than any of the current average dietary lactic acid intake values allowed by several governmental agencies. Thus, PLA is safe for use in fabricating articles for contact with food.

Biocompatible Materials↗

PubMed2002

Detection and false-positive rates of maternal serum markers for Down syndrome screening according to maternal age in women over 35 years of age. A study of the agreement of eight dedicated software packages.

Maternal serum markers for trisomy 21 screening (MSS) can be assayed in women > or =35 years in an attempt to reduce the need for invasive procedures and thereby avoid their side effects. Our objective was to compare, in women > or =35, eight different software packages dedicated to second trimester MSS, thus providing reliable data for patient counselling. A simulation study was carried out on 189 sera from women with Down syndrome fetuses and 11 962 sera from mothers of unaffected babies. The first step was to estimate the joint distribution of alpha-fetoprotein (AFP) and free beta-human chorionic gonadotrophin (beta-hCG). The second step was to calculate trisomy 21 detection and false-positive rates for each software according to maternal age (35-45 years), using the usual 1:250 risk threshold. Agreement between software packages was measured using 2 x 2 kappa coefficients. Detection rates and false-positive rates increased with maternal age. Depending on the software, 57-71% detection rates were achieved at 35 years with 12-18% false-positive rates. At 45 years, 61-100% detection rates were achieved with 66-95% false-positive rates. Up to 39 years, all softwares were concordant (kappa coefficients >0.75). In the range 35-45 years, false-positive and detection rates increased substantially with maternal age and differences between software packages are observed.

Adult↗

PubMed2003

Comparison of PDQuest and Progenesis software packages in the analysis of two-dimensional electrophoresis gels.

Efficient analysis of protein expression by using two-dimensional electrophoresis (2-DE) data relies on the use of automated image processing techniques. The overall success of this research depends critically on the accuracy and the reliability of the analysis software. In addition, the software has a profound effect on the interpretation of the results obtained, and the amount of user intervention demanded during the analysis. The choice of analysis software that best meets specific needs is therefore of interest to the research laboratory. In this paper we compare two advanced analysis software packages, PDQuest and Progenesis. Their evaluation is based on quantitative tests at three different levels of standard 2-DE analysis: spot detection, gel matching and spot quantitation. As test materials we use three gel sets previously used in a similar comparison of Z3 and Melanie, and three sets of gels from our own research. It was observed that the quality of the test gels critically influences the spot detection and gel matching results. Both packages were sensitive to the parameter or filter settings with respect to the tendency of finding true positive and false positive spots. Quantitation results were very accurate for both analysis software packages.

Animals↗

PubMed1999

A physiotherapy test package for assessing back and neck dysfunction--discriminative ability for patients versus healthy control subjects.

BACKGROUND AND PURPOSE: A physiotherapy test package of 10 tests designed for assessing dysfunction due to back or neck pain was tested for its ability to discriminate between back- or neck-pain sufferers and healthy control subjects. Twenty-seven patients and 26 healthy control subjects participated in the study. METHOD: Fifteen patients suffering from back or neck pain and 15 matched healthy control subjects were assessed using the package to compare the results between the groups. The results from a further 12 back- or neck-pain sufferers and 11 unmatched healthy control subjects were analysed with respect to sensitivity and specificity, cluster formation and correlation with ratings of pain and perceived exertion. RESULTS: The differences in the test results for the patients and the matched healthy control subjects were highly significant in all variables but one. High sensitivity for cut-off points of clinical relevance was found in all variables but one, whereas specificity was moderate in most variables. CONCLUSIONS: Despite the small sample size, the significant differences between patients and healthy control subjects, with the sensitivity and specificity figures, suggest that the content validity of the test package is satisfactory.

Adult↗

PubMed2000

Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis.

Apoptosis is characterized by a complex and remarkably ordered choreography of events consisting of the preparatory and execution steps that all culminate in disposal of the cell remnants. The disposal occurs in a manner that is the least destructive to the tissue: the remains of nuclear chromatin and cytoplasm are packaged in apoptotic bodies which are then phagocytized by neighboring live cells without invoking inflammatory or autoimmune response. In the present study we describe that in the course of apoptosis cellular RNA becomes sequestered and packaged into granules and then into apoptotic bodies, separately from DNA. This separation, which appears to be initiated by the nucleolar segregation, was observed in HL-60 cells that were undergoing spontaneous apoptosis in cultures or were treated with the DNA-damaging drug, DNA topoisomerase I inhibitor camptothecin (CPT), or with the cell death ligand, tumor necrosis factor-alpha. RNA separation was also observed in apoptotic MCF-7 cells following treatment with CPT. RNA and DNA in apoptotic cells were identified histochemically, by their differential stainability with pyronin Y and Hoechst 33342 fluorochromes, respectively, and immunocytochemically, by labeling the RNA with BrU for various periods of time and detection of the incorporated precursor with fluoresceinated anti-BrU mAb; DNA was counterstained with 7-aminoactinomycin D. Over 90% of apoptotic bodies that contained RNA had no detectable DNA and vice versa, the apoptotic bodies containing DNA had no detectable RNA. Packaging RNA and DNA into separate apoptotic bodies suggests that the phagosomes of the cells that ingest these particles are specialized: some of them are responsible for DNA degradation, others for degradation of RNA. Such specialization may facilitate heterophagic degradation of nucleic acids during apoptosis.

Apoptosis↗

PubMed1993

The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction.

cosB is the binding site for terminase, the DNA packaging enzyme of ai-12581mbda, and cosN is the adjacent site at which terminase gm-07228es staggered nicks to generate mature lambda DNA molecules. There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase. A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined. Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to congruent to 30% of the level observed for the wild-type substrate. In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the 1 strand and reduced r-strand nicking to 35% of the wild-type level. The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations. Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction. The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed. All of the cosB mutations affect r-strand nicking to a greater extent than 1-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase. The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of terminase for cosB. ATP does accelerate the rate of cos nicking, suggesting that the role of ATP in promoting nicking of the cosB- DNAs is primarily to increase the rate of conversion of a cosN-terminase complex into product. A possible fourth R site, R4, is located on the other side of cosN from cosB.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus.

A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5' end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure-function relationships of the rat VL30 dimerization-encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55 degrees C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5' G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interestingly enough, substitution of an A for a G in the 5' G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possible purine-purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5' G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vector in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5' G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.

3T3 Cells↗

PubMed1997

The bacteriophage phi29 packaging proteins supercoil the DNA ends.

Bacteriophage phi29 DNA with covalently bound terminal protein (DNA-gp3) and its left and right-end restriction fragments (L and R-DNA-gp3) sedimented faster in sucrose density gradients than their proteinase K-treated counterparts, and the faster sedimentation was both gp3 and Mg2+-dependent. Addition of gp16, the phi29 DNA packaging ATPase, further increased the sedimentation rates of both intact DNA-gp3 and L and R-DNA-gp3 fragments. Thus, DNAs with gp3 were more compact than gp3-free DNA, and gp16 further condensed the DNA-gp3 forms. [35S]gp16 cosedimented with the fast-sedimenting DNA-gp3 fragments, and the putative L-DNA-gp3-gp16 complexes were packaged preferentially into proheads in the defined in vitro system. Lariats of DNA-gp3 and L and R-DNA-gp3 observed by electron microscopy rationalized the sedimentation results, and lariats with multiple loops or coils increased tenfold in a preparation of L-DNA-gp3-gp16 complexes. The rapid sedimentation and the structure of the DNA-gp3-gp16 complexes were consistent with supercoiling of lariat loops, and treatment with topoisomerase I shifted fast-sedimenting complexes toward the uncoiled lariat position in sucrose density gradients. DNA-gp3 has a maturation pathway in which the packaging proteins gp3 and gp16 supercoil the DNA ends, probably as a prerequisite for efficient interaction with the prohead.

Adenosine Triphosphatases↗

PubMed1999

Association of holliday-structure resolving endonuclease VII with gp20 from the packaging machine of phage T4.

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the "packasome") located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf-DNA in the absence of Mg2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.

Bacteriophage T4↗

PubMed2000

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform.

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.

Animals↗

PubMed2000

NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition.

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.

Calorimetry↗