A novel antiviral strategy by disrupting the equilibrium of virus-host calcium homeostasis.

Human coronavirus NL63 (HCoV-NL63) is spread globally, causing upper and lower respiratory tract infections mainly in young children. HCoV-NL63 shares a host receptor (ACE2) with severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 but, unlike them, HCoV-NL63 primarily develops into self-limiting mild to moderate respiratory disease. Although with different efficiency, both HCoV-NL63 and SARS-like CoVs infect ciliated respiratory cells using ACE2 as receptor for binding and cell entry. Working with SARS-like CoVs require access to BSL-3 facilities, while HCoV-NL63 research can be performed at BSL-2 laboratories. Thus, HCoV-NL63 could be used as a safer surrogate for comparative studies on receptor dynamics, infectivity and virus replication, disease mechanism, and potential therapeutic interventions against SARS-like CoVs. This prompted us to review the current knowledge on the infection mechanism and replication of HCoV-NL63. Specifically, after a brief overview on the taxonomy, genomic organization and virus structure, this review compiles the current HCoV-NL63-related research in virus entry and replication mechanism, including virus attachment, endocytosis, genome translation, and replication and transcription. Furthermore, we reviewed cumulative knowledge on the susceptibility of different cells to HCoV-NL63 infection in vitro, which is essential for successful virus isolation and propagation, and contribute to address different scientific questions from basic science to the development and assessment of diagnostic tools, and antiviral therapies. Finally, we discussed different antiviral strategies that have been explored to suppress replication of HCoV-NL63, and other related human coronaviruses, by either targeting the virus or enhancing host antiviral mechanisms.

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post‐translational modifications such as covalent conjugation of ubiquitin and small ubiquitin‐related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co‐opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO‐regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.

Typical viral propagation involves sequential viral entry, uncoating, replication, gene transcription and protein synthesis, and virion assembly and release. Some viral proteins must be transported into host nucleus to facilitate viral propagation, which is essential for the production of mature virions. During the transport process, nuclear localization signals (NLSs) play an important role in guiding target proteins into nucleus through the nuclear pore. To date, some classical nuclear localization signals (cNLSs) and non-classical NLSs (ncNLSs) have been identified in a number of viral proteins. These proteins are involved in viral replication, expression regulation of viral genes and virion assembly. Moreover, other proteins are transported into nucleus with unknown mechanisms. This review highlights our current knowledge about the nuclear trafficking of cellular proteins associated with viral propagation.

Viral replication is dependent on a host; with their small genomes, viruses need to hijack host cellular machinery to complete their life cycle. The coevolution of intimate virus-host relationships has led to many viruses being able to successfully propagate without a significant detrimental effect to the host. However, in some instances, viral infection causes disease. The molecular mechanisms underlying virus-associated disease were the focus of an Advanced Summer School in Africa held in Hermanus, South Africa, from 6 to 14 March 2010. Assembled in the coastal town of Hermanus were 45 participants, primarily PhD students and postdocs from less developed countries, and 18 speakers, leaders in their respective fields, from around the world ( Fig. 1). The school, the second of its kind in Africa was sponsored by the International Union of Biochemistry and Molecular Biology (IUBMB), the Federation of European Biochemical Societies (FEBS), the Federation of African Societies of Biochemistry and Molecular Biology, the United Nations Educational Scientific and Cultural Organization, and the International Centre for Genetic Engineering and Biotechnology (ICGEB). The course was honored by the presence of Brian Clark, Professor of Biostructural Chemistry at The University of Aarhus, Denmark. Prof Clark is a renowned scientist who participated in the discovery of the initiation codon for protein synthesis (1-4) and the first crystallization of a tRNA (5). Today, Prof Clark studies the molecular and cellular mechanisms of ageing using a systems biology approach. His presence was particularly significant at the Summer School since he has been involved in the organization of several similar events during his tenure as President of the IUBMB and FEBS, most notably the Spetses Summer School. Prof Clark stressed the importance of such meetings for the development, not just of young scientists, but of science itself. Participants of the Second Advancement Summer School in Africa. 45 young researchers and 18 speakers were assembled in the beautiful coastal town of Hermanus, South Africa. The main themes of discussion at the summer school were: 1) why viral infection can lead to cancer; 2) how a greater understanding of the mechanisms underpinning human immunodeficiency virus (HIV) propagation can inform new antiviral strategies; 3) the abilities of viruses to evade the immune system and the obstacles to the development of effective vaccines; and, 4) the potential afforded by viruses as research tools. The importance of host factors became apparent in the discussion of all these topics, and how viral research has informed our general knowledge of cell biology. This report serves to summarise the findings presented at the summer school. Until Peyton Rous discovered the first tumor virus in 1911, viruses were viewed as peculiar infectious agents capable of inducing cancer in animals, but not in humans. However, it is now well-established that many different human viruses, including Human papilloma virus (HPV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), Human T-cell leukemia virus-1 (HTLV-1), Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Merkel cell polyoma virus (MCV) are the etiological agents of human cancers, encompassing at least 15–20% of all tumors worldwide (6). Hence, tumor virology was a focus of this summer school. Dr Lawrence Banks from the ICGEB, Trieste, provided an overview of HPV and the role of viral proteins in the transformation of cervical epithelial cells. Almost all (99.7%) cervical cancers contain HPV DNA, usually types 16 and 18 (7). Despite this statistic, cancer induction is not part of the 'normal' HPV life cycle. Only in rare cases where there has been a lack of immune clearance leading to persistent infection, along with additional changes in the cell, does cancer develop. Banks described how HPV E6 and E7 are the major viral oncoproteins involved in the development of cervical cancer. The HPV life cycle is critically dependent on the differentiation of epithelial cells: there is usually no DNA replication, other than in cells of the basement layer, but HPV E6 and E7 induce a pseudo-S phase, creating a permissive environment for viral replication (8). In addition, E6 and E7 target cellular substrates for proteasome-mediated degradation, notably the tumor suppressors p53 and pRb, respectively (9, 10). HPV DNA exists in an episomal form but may be found integrated in malignant cells. Integration often involves loss of viral DNA. Indeed, malignant cells often display no viral replication but maintain expression of E6 and E7 (11), further emphasizing the roles of these proteins in cancer progression. But why do only a few HPV infections result in malignancy? This issue was tackled by Dr John Doorbar, National Institute for Medical Research, London. HPV-associated cancers are more predominant in some tissues than others: there are ∼500,000 cases of cervical cancer reported each year. In contrast, HPV-associated penile cancers are rare (∼40,000 cases per year), despite no evidence of a disparity in primary infection rates. This implies a difference in the propensity of HPV to form neoplastic lesions at different sites. Doorbar speculates that this is due to a graded ability of HPV to replicate in different tissues. As further evidence, HPV-1 causes warts but no lesions at mucosal sites. Doorbar believes that the transformation zone in the cervix may be particularly susceptible to neoplasia formation. The exact host factors responsible for the variation in HPV replication ability are unresolved. Doorbar's group is also investigating the molecular basis of the different outcomes resulting from HPV infection. HPV gene expression occurs in an ordered manner as the infected cells move through the epithelial tissue. Doorbar described how the host protein mini chromosome maintenance protein 7 (MCM7) could be used as a marker for cell proliferation and, thus, as a surrogate for E6/7 activity. MCM7 was expressed in the lower layers of HPV lesions, as shown by laser capture microscopy, and the viral protein E4 was abundantly expressed in the upper layers (12). Interestingly, Doorbar's group have shown that high-grade squamous intraepithelial lesions (HSILs) are associated with increased MCM7 expression in upper epithelial layers with almost no E4 expression (12), revealing that viral expression patterns reflect the severity of disease. In addition, proliferation of the basal cells does not stop when the basement layer is complete in high-risk lesions, in contrast to low-risk lesions. The increased basal layer proliferation is correlated with beta-catenin activity and E6 levels (13, 14). What drives increased E6 activity in some infections is unknown: wound healing, episomal copy number, DNA methylation patterns, and host genetic background could all contribute and require further investigation. Nevertheless, these observations have the potential to tremendously improve existing cervical screening practice, which relies heavily on cytology. Though in vitro studies are invaluable for identifying candidate molecules involved in pathogenesis, conformation of their role often requires an in vivo approach. Dr Paul F. Lambert, University of Wisconsin Medical School, described transgenic mouse models of HPV where the human keratin promoter, hK14, was used to drive expression of HPV genes in stratified squamous epithelia (15). Previous studies had shown that expression of HPV genes was insufficient to cause cervical cancer in mice (15). Lambert's group hypothesized that estrogen was a cofactor for cervical cancer. They generated hK14-HPV16-estrogen receptor transgenic mice, which did develop cervical cancer (16-18). This may account for observations that >5 years of contraceptive pill usage and pregnancy increases the risk of women developing cervical cancer. HPV is not just associated with cancer of the cervix, however. Prof Iqbal Parker of the ICGEB, Cape Town, showed that papilloma viruses typically considered low-risk could be involved in fatal cancers of the oesophagus. Parker's group found that about 40% of oesophageal cancer (OC) patients had integrated HPV-11 and -39 DNA in their tumors (19). Brush biopsies of healthy people confirmed that HPV was enriched in OC patients. However, smoking, alcoholic home brew consumption and cooking on indoor fires in conjunction with certain Sulpho-transferase alleles were also risk factors associated with the development of OC in Africa (20). Whether HPV infection is an early or initiation event in OC development requires further investigation. The molecular alterations that direct progression from productive infection to HSILs in HPV infection are not fully understood. HPV oncoproteins alone are not capable of transformation, as shown in human keratinocytes in vitro (21) and murine cervical epithelia in vivo (15). Rather, papilloma virus-mediated oncogenesis requires supplementary genetic changes that occur over time following the initial infection. Whilst it is clear that integration of viral DNA into the host genome is crucial to HPV-induced tumor development (22, 23), whether this is a cause or consequence of wider-spread chromosome instability is not clear. Despite the activity of various oncogenic HPV proteins, viral DNA integration and cancer development does not often follow. The import of the host, including an ability to regulate viral gene expression in different tissues and to mount an effective immune response, is becoming increasingly apparent in determining the molecular basis of HPV-associated tumor progression. Dr Ethel Cesarman, Weill Cornell Medical College, described Human Herpes viruses and focused on KSHV, also known as Human Herpes Virus 8 (HHV8). KSHV causes a cancer often found in immunosuppressed individuals, such as those suffering from AIDS, including Karposi's sarcoma primary effusion lymphoma and multicentric Castleman's disease. There is >50% KSHV seroprevalence in sub-Saharan Africa but, as with HPV, the majority of individuals present with no disease. KSHV contains up to 90 ORF with at least 15 accessory genes. Among the accessory genes identified to date, ORF74, named KSHV G protein-coupled receptor (KSHV-GPCR), has become a major focus of investigation. Though it is expressed in a very small number of KS lesion spindle cells (24), it is known to be involved in cell transformation through induction of angiogenic cytokine expression (25). For example, vascular endothelial growth factor is one cytokine secreted from cells expressing KSHV-GPCR, which can induce proliferation and angiogenesis in KS tissue (26). Following the observation that proteins expressed by lymphomagenic viruses during latent infection activate NF-κB, Cesarman's group searched for a viral protein whose activity correlated with NF-κB. They identified vFLIP, an homologue of cFLIP proteins, and showed that anti-vFLIP siRNAs induced apoptosis in KSHV-positive cells only (27). Work is continuing to develop drugs that are vFLIP-specific, in the hope that these will prove effective anti-KSHV agents. During his lectures, Dr Ingemar Ernberg, of the Karolinska Institute in Stockholm, recoined the acronym EBV as "Every Bodies Virus" because >95% of humans are infected from childhood, though infections are typically subclinical. A member of the HHV family, EBV was initially isolated from a Burkitt's lymphoma cell line (28), though it was later found associated with a number of different tumors, such as undifferentiated nasopharyngeal carcinoma, Hodgkin's disease, nasal T cell lymphoma and gastric carcinoma. Ernberg described the four programs of latent viral gene expression (L0–L3) and how all 12 viral genes are expressed in L3, which is associated with cell proliferation (29). Therefore, Ernberg's group looked for the signal controlling the switch from L1 to L3. They showed that in L1, there was low expression of the viral gene EBNA1, but high levels of the cellular transcription factor Oct2, and vice versa in L3. Work is ongoing into deciphering the reason for a decrease in Oct2 levels that is concomitant with a switch to the L3 program of gene expression. Hopefully this will shed light on why EBV set points vary between individuals and, further, what differs between the B cells of those who appear immune to EBV infection and the majority of the population who are susceptible. Dr Aluisio Segurado, University of São Paulo, described HTLV-1, a retrovirus that can result in Adult T cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM/TSP). It is estimated that 15 to 20 million individuals are infected with HTLV-1 worldwide, yet the vast majority remain clinically asymptomatic--only 2–6% develop ATLL (30). The reason for the low frequency of HTLV-1-associated disease is unknown but Segurado hypothesises that host genetics and environmental factors play a role, rather than infection with different strains of HTLV. Tax (p40tax) is one of the HTLV-1 proteins that acts as a transcription activator in ATLL (31). However, the low incidence rate and the long latency period prior to development of ATLL suggests that, in addition to viral infection, accumulation of mutations in host genes is required for cellular transformation in vivo. Among the hepatitis viruses, HBV and HCV cause chronic infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC). HBV is a DNA virus that integrates into the host genome. Dr Shahid Jameel, ICGEB, New Delhi, described how this integration deregulates expression of the viral protein HBx, which is able to induce HCC, either alone or in synergy with different cellular proteins (32). Jameel also discussed tumorigenesis induced by HCV: chronic immune-mediated inflammation and associated oxidative chromosomal DNA damage probably play a role. Interactions of viral proteins with pRb and p53 may also predispose to carcinogenesis (33). Although not all the viruses discussed induce the same pathway to cancer, some parallels can be drawn. A requirement for viral oncogenes indicates that the viruses play a crucial role in progression to malignancy. Both RNA and DNA tumor viruses promote growth of infected tissue by activation of cellular oncogenes and inactivation of tumor suppressor genes. Interestingly, many cellular oncogenes and tumor suppressor genes (e.g., p53, pRb) were identified through studies of RNA and DNA tumor viruses, respectively. After infection, these viruses can establish either latent (HPV, EBV, KSHV, HTLV-1) or chronic (HBV, HCV) infection. Activation from latent infection may result from changes in the cell environment, an accumulation of viral stress on the cell and/or a decreased ability of the host to maintain latency or clear infection. Such activation signals may be dependent on the host's genetic make-up (34) and epigenetic factors (35). Understanding why some individuals infected with tumor viruses do not develop cancer remains critical. It is anticipated that continuing research into what distinguishes 'normal' viral life cycles from the life cycles in malignant tissues, and the changes in host factors accompanying transformation, will address this problem. So far it has been demonstrated that changes in viral gene expression may accompany the development of high-risk lesions (e.g., HPV, EBV). But why are such changes occurring at a low frequency in infected populations? Are these changes in gene expression a result of a virus-host interaction disruption? Answering these questions requires a great deal more research, but certainly the speakers at the Summer School are on course to do just that. A detailed understanding of the molecular mechanisms governing virus infection and propagation is crucial to the development of antiviral strategies through identification of critical processes and drug targets. This was best exemplified at the Summer School by three speakers who are elucidating complimentary stages of the HIV life cycle: Ariberto Fassati, University College London, discussed nuclear import, Alessandro Marcello, ICGEB, Trieste, focused on integration and the spatial and temporal regulation of HIV-1 gene expression, and Hans-Georg Kräusslich, Universität Heidelberg, dealt with assembly and maturation. Although existing highly active antiretroviral therapy (HAART) can significantly reduce HIV-related illness, the emergence of drug resistant strains, toxic side effects and their ineffectiveness in latently infected cells has ensured that the development of novel HIV-1 therapies remains an important objective. Lentiviruses, such as HIV-1, have the ability to infect terminally differentiated nondividing cells and, therefore, require nuclear import. However, the HIV-1 reverse transcription complex (RTC) is larger than the nuclear pore diffusion size limit. In addition, the RTC is enriched for nucleic acids and so must overcome the hydrophobic exclusion of nuclear pore complexes and a concentration gradient of DNA to enter the nucleus. The manner in which HIV-1 resolves this dilemma is worth investigating as nuclear import is critical to HIV-1 transmission and AIDS pathogenesis. Dr A. Fassati is doing just that and his group has identified some novel mechanisms by which HIV-1 enters the nucleus. A number of viral elements have been shown to play a role in nuclear import e.g., the cPPT element, a nuclear localization signal within matrix and the viral proteins integrase (IN) and Vpr. Fassati's group was interested in identifying the host nuclear transport receptor responsible for HIV-1 import. They used purified RTC complexes in primary macrophages to show that importin 7 (imp7) is involved (36), an import receptor for RNA- and DNA-binding ribosomal and histone proteins, respectively. Further work revealed that the role of imp7 was HIV-specific, which correlated with its ability to bind HIV IN (37). RNAi-mediated knockdown of imp7 decreased import of DNA, but not RNA, indicating that reverse transcription is not a requirement for nuclear import (37). The DNA import-function of imp7 is likely hijacked by HIV-1 to facilitate import of the HIV-1 DNA genome, although it remains unclear whether the RTC remains intact throughout the nuclear import process. Fassati's group continued to search for other nuclear import pathways. They used nucleic acid dye-labeled RTCs and cells treated with digitonin (to permeabilise the plasma, but not the nuclear, membrane) to monitor nuclear import in the presence of various cytosolic extracts. The almost capable of inducing nuclear import of HIV-1 RTCs was found to contain showed that nuclear import of at least some of occurs in cells cellular pathway of unknown that HIV-1 to are present in HIV-1 and it is that the nuclear import of the RTC with host which have yet to be Fassati's work not only novel import for HIV-1, and thus, potential drug it has also our knowledge of cellular biology through identification of the tRNA nuclear import viral are early during HIV infection, which of the as are not susceptible to antiretroviral In addition, latent DNA can be to viral on of Therefore, understanding the molecular mechanisms governing latency and of viral expression is crucial to developing strategies at complete of the this Dr A. described his work into the of and chromosome on the of HIV-1 transcription and has shown that the histone the protein and histone is enriched at DNA can the on HIV-1 gene expression in from Whilst into the mechanisms underlying HIV-1 the remains as to how some cells to be latently with at the the propensity for HIV-1 to into active The is a highly and and it is now that active transcription is enriched at the nuclear group the latently infected cell cells developed by by and showed that along with viral transcription occurring at the nuclear the latent was at the conformation capture revealed an of the latent with a of chromosome 12 in which was on of transcription Although it be that latency is a result of a rare integration event into an gene at the nuclear work a in which integration into an active gene and of the T cell population is by a number of T cells becoming cells by a that is by the of the through epigenetic of this in an manner is an ongoing The host cell is a environment, so need to form in the but the to be into a new viruses have different to this In the is provided by the a with a of is During of the by in a of the to form a infectious virus with a of the of despite no in the size of the Dr provided an overview of his work the mechanisms of virus and the to studies revealed that following of the contains proteins assembled into a A was to contain more than the number present in the studies demonstrated that, in is in an in addition, contains The may to the by the as there is no evidence of as in the The of and its in the and stages may a of the prior to assembly of the The of is but such a be an drug the virus contains an how is the virus from the it was that assembled at the into that in with of the resulting by the cellular complex required for transport machinery group demonstrated that almost complete were only present at where was with their observations that the is of an a novel for the of HIV-1 is by assembly with early to drive The can therefore, considered a assembly occur than is microscopy, group was able to that an in at of HIV-1 early in assembly understanding of the role of host in HIV-1 to be an HIV-1 is by the HIV accessory protein However, and mouse HIV-1 through its to This has for the development of small models of HIV infection. The work of Kräusslich, Marcello, and Fassati that continued into the molecular mechanisms of HIV-1 replication development of effective an to all The immune system is important in the detrimental effects of HPV more lesions are in patients of HPV DNA in women is which may be due to changes in the immune Dr Doorbar described how the immune system to clear cells are not very effective as viral proteins are expressed at low levels in the lower epithelial In addition, HPV proteins E6 and E7 and respectively is also to play an role through of major Hence, without HPV-associated warts and can to It is also this in immune clearance of the virus that may predispose individuals to cancer. an HPV is Whilst the only the most predominant high-risk so that it is Nevertheless, of this reduce the cervical cancer of has the world over Prof the immune to this humans successfully following the of although the virus is to prior to The of a is of for a gene to a virus that is able to infect humans and cause to the protein are However, the virus is able to the immune system through and the the potential to cause of to virus infection in mice, but of did not This the that requires not which to be in also discussed the of to the antiviral immune described the in which with one and with not just but also to For example, with will not e.g., but will improve the primary to years This to the of B cells as and cells: T B cell not just B cell following primary to T cell a more response, including to This is important in as it the importance of not just the of but also the so as to was first reported in in and within a few the virus had to more than in South and Prof a detailed on his development of a His group has demonstrated that virus mice from fatal disease when with and mice expressing the viral with showed decreased inflammation and on The also induced T cell and Work is ongoing to the role of but gene expression studies regulation of the stress Whilst highly the is not yet considered for particularly the of and the risk of involves but this also as inactivation can be The of rather than in could improve the of without HIV has infected more than million people

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication.

More than 60% of plant viruses are positive-strand RNA viruses that cause billion-dollar losses annually and pose a major threat to stable agricultural production, including cucumber mosaic virus (CMV) that infects numerous vegetables and ornamental trees. A highly conserved feature among these viruses is that they form viral replication complexes (VRCs) to multiply their genomes by hijacking host proteins and remodeling host intracellular membranes. As a conserved and indispensable process, VRC assembly also represents an excellent target for the development of antiviral strategies that can be used to control a wide-range of viruses. Using CMV and a model virus, brome mosaic virus (BMV), and relying on genomic tools and tailor-made large-scale resources specific for the project, our original objectives were to: 1) Identify host proteins that are required for viral replication complex assembly. 2) Dissect host requirements that determine viral host range. 3) Provide proof-of-concept evidence of a viral control strategy by blocking the viral replication complex-localized phospholipid synthesis. We expect to provide new ways and new concepts to control multiple viruses by targeting a conserved feature among positive-strand RNA viruses based on our results. Our work is going according to the expected timeline and we are progressing well on all aims. For Objective 1, among ~6,000 yeast genes, we have identified 96 hits that were possibly play critical roles in viral replication. These hits are involved in cellular pathways of 1) Phospholipid synthesis; 2) Membrane-shaping; 3) Sterol synthesis and transport; 4) Protein transport; 5) Protein modification, among many others. We are pursuing several genes involved in lipid metabolism and transport because cellular membranes are primarily composed of lipids and lipid compositional changes affect VRC formation and functions. For Objective 2, we have found that CPR5 proteins from monocotyledon plants promoted BMV replication while those from dicotyledon plants inhibited it, providing direct evidence that CPR5 protein determines the host range of BMV. We are currently examining the mechanisms by which dicot CPR5 genes inhibit BMV replication and expressing the dicot CPR5 genes in monocot plants to control BMV infection. For Objective 3, we have demonstrated that substitutions in a host gene involved in lipid synthesis, CHO2, prevented the VRC formation by directing BMV replication protein 1a (BMV 1a), which remodels the nuclear membrane to form VRCs, away from the nuclear membrane, and thus, no VRCs were formed. This has been reported in Journal of Biological Chemistry. Based on the results from Objective 3, we have extended our plan to demonstrate that an amphipathic alpha-helix in BMV 1a is necessary and sufficient to target BMV 1a to the nuclear membrane. We further found that the counterparts of the BMV 1a helix from a group of viruses in the alphavirus-like superfamily, such as CMV, hepatitis E virus, and Rubella virus, are sufficient to target VRCs to the designated membranes, revealing a conserved feature among the superfamily. A joint manuscript describing these exciting results and authored by the two labs will be submitted shortly. We have also successfully set up systems in tomato plants: 1) to efficiently knock down gene expression via virus-induced gene silencing so we could test effects of lacking a host gene(s) on CMV replication; 2) to overexpress any gene transiently from a mild virus (potato virus X) so we could test effects of the overexpressed gene(s) on CMV replication. In summary, we have made promising progress in all three Objectives. We have identified multiple new host proteins that are involved in VRC formation and may serve as good targets to develop antiviral strategies; have confirmed that CPR5 from dicot plants inhibited viral infection and are generating BMV-resistance rice and wheat crops by overexpressing dicot CPR5 genes; have demonstrated to block viral replication by preventing viral replication protein from targeting to the designated organelle membranes for the VRC formation and this concept can be further employed for virus control. We are grateful to BARD funding and are excited to carry on this project in collaboration.

The accurate simulation of realistic biomembranes is a long-term goal in the field of membrane biophysics. Efforts to simulate increasingly complex lipid bilayers, consisting of multiple lipid types and proteins, have been hindered by the shortcomings of current force fields, both coarse-grained and all-atom, in the modeling of protein-protein and protein-lipid interactions. Due to the fundamental importance of protein dimerization to cellular signaling and protein trafficking, the study of protein-protein association and the related dimerization free energies has received significant attention in both simulations and experiments. Detailed comparisons of simulation results with NMR, crystallography, and FRET studies served as a test of the accuracy of the simulation methods and provided insights into the underlying structural distributions and thermodynamic driving forces defining the interactions. These comparisons have led to the conclusion that existing state-of-the-art simulation methods have failed to effectively sample the equilibrium between associated and dissociated states, leading to inaccurate estimates of binding constants and misrepresentation of the associated structural ensembles. Here, we discuss the drawbacks of previously used protocols and review our systematic development of effective computational methods for enhanced sampling simulations that exhaustively sample the native and non-native dimer conformations and provide precise estimates of the associated equilibrium binding constants. We conclude by identifying the most important current challenges to the field that must be met in closing the gap between simulation and experiment in the study of protein-protein association in the membrane.

Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer's disease

Lipid-Anchored Ras is Sorted by Membrane Curvature Both In Vitro and in Living Cells

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.

IntroductionHigh grade serous ovarian carcinoma (HGSOC) is the leading cause of death among all gynecological malignancies. Extracellular microvesicles (MVs) are secreted by most cells in the body and play a...

IntroductionChronic amphetamine use causes profound adaptations in the brain including desensitization and sensitization of many GPCRs and altered signaling of G proteins coupled to these receptors. These changes are associated with vulnerability to relapse. However, the mechanisms underlying changes in receptor‐mediated G protein signaling remain elusive.One possible mechanism is an alteration in the compartmentalization of G proteins in the plasma membrane. Lipid rafts are the subcompartment of the plasma membranes characterized by high cholesterol content. They act as microdomains to accommodate cellular signaling and protein trafficking. Changes in the lipid composition of the plasma membrane would alter protein localization and associated signaling. Little is known about the effects of psychostimulant treatments on G protein distribution in lipid raft and nonraft domains of striatal cell membranes.The purpose of this study was to use an animal model of amphetamine self‐administration to investigate the parallel changes in the brain cholesterol content and G protein membrane compartmentalization. This study may shed light on the contribution of membrane cholesterol content to amphetamine‐induced alteration in G protein signaling and receptor responses to stimuli.MethodsMale Sprague Dawley rats were anesthetized, implanted with catheters, and allowed to self‐administer amphetamine (0.189 mg/kg/infusion) on a fixed ratio one schedule. Animals were allowed to self‐administer up to 40 injections in 6 hrs per day over 5 or 14 days. Rats were sacrificed and the striata were dissected. Striatal tissues were fractionated using a sucrose density gradient and centrifugation to isolate lipid raft and non‐lipid raft fractions. Fractions were subjected to immunoblotting to determine G protein localization. Amplex Red Hydrogen Peroxide/Peroxidase assay kit was used to measure the striatal cholesterol levels.ResultsOur data showed both 5 and 14 days of amphetamine self‐administration significantly reduced the total cholesterol levels in the striatum. Interestingly, we found that amphetamine self‐administration differentially regulated the membrane compartmentalization of Gα subtypes. For Gαo and Gαs, there was a notable translocation from nonraft to raft fractions in both 5 and 14 days amphetamine self‐administering rats. For Gαi2 and Gαi3, there was no change in membrane compartmentalization following 5 days of amphetamine self‐administration; however, 14 days of amphetamine exposure caused these G‐proteins to translocate from raft to nonraft fractions. No significant membrane translocation of Gαq was observed. This study provides first time evidence that amphetamine self‐administration selectively alters the membrane compartmentalization of Gα subunits, which may be associated with changes in receptor function and addiction‐like behavior. Future research on the contribution of the membrane cholesterol content to receptor function and addiction behavior is warranted.Support or Funding InformationThis project is supported by NIH DA006634.

HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

Simvastatin inhibits protein isoprenylation in the brain

Influenza A viral ribonucleoprotein (vRNP) is responsible for transcription and replication of the viral genome in infected cells and depends on host factors for its functions. Identification of the host factors interacting with vRNP not only improves understanding of virus-host interactions but also provides insights into novel mechanisms of viral pathogenicity and the development of new antiviral strategies. Here, we have identified 80 host factors that copurified with vRNP using affinity purification followed by mass spectrometry. LYAR, a cell growth-regulating nucleolar protein, has been shown to be important for influenza A virus replication. During influenza A virus infection, LYAR expression is increased and partly translocates from the nucleolus to the nucleoplasm and cytoplasm. Furthermore, LYAR interacts with RNP subunits, resulting in enhancing viral RNP assembly, thereby facilitating viral RNA synthesis. Taken together, our studies identify a novel vRNP binding host partner important for influenza A virus replication and further reveal the mechanism of LYAR regulating influenza A viral RNA synthesis by facilitating viral RNP assembly.IMPORTANCE Influenza A virus (IAV) must utilize the host cell machinery to replicate, but many of the mechanisms of IAV-host interaction remain poorly understood. Improved understanding of interactions between host factors and vRNP not only increases our basic knowledge of the molecular mechanisms of virus replication and pathogenicity but also provides insights into possible novel antiviral targets that are necessary due to the widespread emergence of drug-resistant IAV strains. Here, we have identified LYAR, a cell growth-regulating nucleolar protein, which interacts with viral RNP components and is important for efficient replication of IAVs and whose role in the IAV life cycle has never been reported. In addition, we further reveal the role of LYAR in viral RNA synthesis. Our results extend and improve current knowledge on the mechanisms of IAV transcription and replication.