Nuclear Functions of Nucleolin through Global Proteomics and Interactomic Approaches

International audienceNucleolin (NCL) is a major component of the cell nucleolus, which has the ability to rapidly shuttle to several other cells' compartments. NCL plays important roles in a variety of essential functions, among which are ribosome biogenesis, gene expression, and cell growth. However, the precise mechanisms underlying NCL functions are still unclear. Our study aimed to provide new information on NCL functions via the identification of its nuclear interacting partners. Using an interactomics approach, we identified 140 proteins co-purified with NCL, among which 100 of them were specifically found to be associated with NCL after RNase digestion. The functional classification of these proteins confirmed the prominent role of NCL in ribosome biogenesis and additionally revealed the possible involvement of nuclear NCL in several pre-mRNA processing pathways through its interaction with RNA helicases and proteins participating in pre-mRNA splicing, transport, or stability. NCL knockdown experiments revealed that NCL regulates the localization of EXOSC10 and the amount of ZC3HAV1, two components of the RNA exosome, further suggesting its involvement in the control of mRNA stability. Altogether, this study describes the first nuclear interactome of human NCL and provides the basis for further understanding the mechanisms underlying the essential functions of this nucleolar protei

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments▿

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus

Nucleolin interacts with US11 protein of herpes simplex virus 1 and is involved in its trafficking.

International audienceHerpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection

Analysis of <i>Mycobacterium tuberculosis</i> Genotypic Lineage Distribution in Chile and Neighboring Countries

<div><p>Tuberculosis (TB), caused by the pathogen <i>Mycobacterium tuberculosis</i> (MTB), remains a disease of high importance to global public health. Studies into the population structure of MTB have become vital to monitoring possible outbreaks and also to develop strategies regarding disease control. Although Chile has a low incidence of MTB, the current rates of migration have the potential to change this scenario. We collected and analyzed a total of 458 <i>M</i>. <i>tuberculosis</i> isolates (1 isolate per patient) originating from all 15 regions of Chile. The isolates were genotyped using the spoligotyping method and the data obtained were analyzed and compared with the SITVIT2 database. A total of 169 different patterns were identified, of which, 119 patterns (408 strains) corresponded to Spoligotype International Types (SITs) and 50 patterns corresponded to orphan strains. The most abundantly represented SITs/lineages were: SIT53/T1 (11.57%), SIT33/LAM3 (9.6%), SIT42/LAM9 (9.39%), SIT50/H3 (5.9%), SIT37/T3 (5%); analysis of the spoligotyping minimum spanning tree as well as spoligoforest were suggestive of a recent expansion of SIT42, SIT50 and SIT37; all of which potentially evolved from SIT53. The most abundantly represented lineages were LAM (40.6%), T (34.1%) and Haarlem (13.5%). LAM was more prevalent in the Santiago (43.6%) and Concepción (44.1%) isolates, rather than the Iquique (29.4%) strains. The proportion of X lineage was appreciably higher in Iquique and Concepción (11.7% in both) as compared to Santiago (1.6%). Global analysis of MTB lineage distribution in Chile versus neighboring countries showed that evolutionary recent lineages (LAM, T and Haarlem) accounted together for 88.2% of isolates in Chile, a pattern which mirrored MTB lineage distribution in neighboring countries (n = 7378 isolates recorded in SITVIT2 database for Peru, Brazil, Paraguay, and Argentina; and published studies), highlighting epidemiological advantage of Euro-American lineages in this region. Finally, we also observed exclusive emergence of patterns SIT4014/X1 and SIT4015 (unknown lineage signature) that have hitherto been found exclusively in Chile, indicating that conditions specific to Chile, along with the unique genetic makeup of the Chilean population, might have allowed for a possible co-evolution leading to the success of these emerging genotypes.</p></div

Description of clusters containing >1% (n = 5 or more isolates) in this study, and their worldwide distribution in the SITVIT2 database.

<p>Description of clusters containing >1% (n = 5 or more isolates) in this study, and their worldwide distribution in the SITVIT2 database.</p

Spoligoforest tree based on all spoligotypes (n = 458 isolates).

<p>Spoligoforest was drawn using the Fruchterman-Reingold algorithm from the SpolTools software (<a href="http://www.emi.unsw.edu.au/spolTools" target="_blank">http://www.emi.unsw.edu.au/spolTools</a>)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160434#pone.0160434.ref029" target="_blank">29</a>], and reshaped and colored using the GraphViz software (<a href="http://www.graphviz.org/" target="_blank">http://www.graphviz.org</a>)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160434#pone.0160434.ref030" target="_blank">30</a>]. Each spoligotype pattern from the study is represented by a node with area size being proportional to the total number of isolates with that specific pattern. Changes (loss of spacers) are represented by directed edges between nodes, with the arrowheads pointing to descendant spoligotypes. The heuristic used selects a single inbound edge with a maximum weight using a Zipf model. Solid black lines link patterns that are very similar, i.e., loss of one spacer only (maximum weigh being 1.0), while dashed lines represent links of weight comprised between 0.5 and 1, and dotted lines a weight less than 0.5.</p

Description of 119 shared-types (SITs; n = 408 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 458 <i>M</i>. <i>tuberculosis</i> strains isolated in various cities in Chile.

<p>Description of 119 shared-types (SITs; n = 408 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 458 <i>M</i>. <i>tuberculosis</i> strains isolated in various cities in Chile.</p

A comparison of the proportion of predominant SITs found in Chile (n = 458 isolates in total, as well as distribution patterns for major cities, i.e., Santiago, Iquique, and Concepción), as compared to neighboring countries (Peru, Brazil, Paraguay, Argentina), recorded in the SITVIT2 database.

<p>A comparison of the proportion of predominant SITs found in Chile (n = 458 isolates in total, as well as distribution patterns for major cities, i.e., Santiago, Iquique, and Concepción), as compared to neighboring countries (Peru, Brazil, Paraguay, Argentina), recorded in the SITVIT2 database.</p

Phylogeographical distribution of <i>M</i>. <i>tuberculosis</i> lineages identified in our study and MST illustrating evolutionary relationships.

<p>(A) Phylogeographical distribution of major <i>M</i>. <i>tuberculosis</i> lineages in the 3 most important cities in our study (Santiago, Iquique, Concepción) as well as following neighboring countries: Peru, Brazil, and Paraguay (referring to the SITVIT2 database). (B) A minimum spanning tree (MST) illustrating evolutionary relationships between spoligotypes in various cities of Chile (n = 458 isolates). The phylogenetic tree connects each genotype based on degree of changes required to go from one allele to another. The structure of the tree is represented by branches (continuous vs. dashed and dotted lines) and circles representing each individual pattern. Note that the length of the branches represents the distance between patterns while the complexity of the lines (continuous, gray dashed and gray dotted) denotes the number of allele/spacer changes between two patterns: solid lines, 1 or 2 or 3 changes (thicker ones indicate a single change, while the thinner ones indicate 2 or 3 changes); gray dashed lines represent 4 changes; and gray dotted lines represent 5 or more changes. The size of the circle is proportional to the total number of isolates in our study, illustrating unique isolates (smaller nodes) versus clustered isolates (bigger nodes). The color of the circles indicates the phylogenetic lineage to which the specific pattern belongs. The labels of nodes indicate predominant SITs in study (containing at least 5 or more isolates).</p