Viral Replication Process Research Articles

Viral and cellular interactions during adenovirus DNA replication.

Adenoviruses represent ubiquitous and clinically significant human pathogens, gene-delivery vectors, and oncolytic agents. The study of adenovirus-infected cells has long been used as an excellent model to investigate fundamental aspects of both DNA virus infection and cellular biology. While many key details supporting a well-established model of adenovirus replication have been elucidated over a period spanning several decades, more recent findings suggest that we have only started to appreciate the complex interplay between viral genome replication and cellular processes. Here, we present a concise overview of adenovirus DNA replication, including the biochemical process of replication, the spatial organization of replication within the host cell nucleus, and insights into the complex plethora of virus-host interactions that influence viral genome replication. Finally, we identify emerging areas of research relating to the replication of adenovirus genomes.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling

The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.

Mathematical modeling and analysis of innate and humoral immune responses to dengue infections

Dengue is an acute arthropode-borne virus, belonging to the family Flaviviridae. Currently, there are no vaccines or treatments available against dengue. Thus it is important to understand the dynamics of dengue in order to control the infection. In this paper, we study the long-term dynamics of the model that is presented in [S. D. Perera and S. S. N. Perera, Simulation model for dynamics of dengue with innate and humoral immune responses, Comput. Math. Methods Med. 2018 (2018) 8798057, 18 pp. https://doi.org/10.1155/2018/8798057 ] which describes the interaction of virus with infected and uninfected cells in the presence of innate and humoral immune responses. It was found the model has three equilibria, namely: infection free equilibrium, no immune equilibrium and endemic equilibrium, then analyzed its stability analytically. The analytical findings of each model have been exemplified by numerical simulations. Given the fact that intensity of dengue virus replication at early times of infection could determine clinical outcomes, it is important to understand the impact of innate immunity, which is believed to be the first line of defense against an invading pathogen. For this we carry out a simulation case study to investigate the importance of innate immune response on dengue virus dynamics. A comparison was done assuming that innate immunity was active; innate immunity was in quasi-steady state and innate immunity was inactive during the virus replication process. By a further analysis of the qualitative behavior of the quasi-steady state, it was observed that innate immune response plays a pivotal role in dengue virus dynamics. It can change the dynamical behavior of the system and is essential for the virus clearance.

JCPyV-Induced MAPK Signaling Activates Transcription Factors during Infection.

JC polyomavirus (JCPyV), a ubiquitous human pathogen, is the etiological agent of the fatal neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Like most viruses, JCPyV infection requires the activation of host-cell signaling pathways in order to promote viral replication processes. Previous works have established the necessity of the extracellular signal-regulated kinase (ERK), the terminal core kinase of the mitogen-activated protein kinase (MAPK) cascade (MAPK-ERK) for facilitating transcription of the JCPyV genome. However, the underlying mechanisms by which the MAPK-ERK pathway becomes activated and induces viral transcription are poorly understood. Treatment of cells with siRNAs specific for Raf and MAP kinase kinase (MEK) targets proteins in the MAPK-ERK cascade, significantly reducing JCPyV infection. MEK, the dual-specificity kinase responsible for the phosphorylation of ERK, is phosphorylated at times congruent with early events in the virus infectious cycle. Moreover, a MAPK-specific signaling array revealed that transcription factors downstream of the MAPK cascade, including cMyc and SMAD4, are upregulated within infected cells. Confocal microscopy analysis demonstrated that cMyc and SMAD4 shuttle to the nucleus during infection, and nuclear localization is reduced when ERK is inhibited. These findings suggest that JCPyV induction of the MAPK-ERK pathway is mediated by Raf and MEK and leads to the activation of downstream transcription factors during infection. This study further defines the role of the MAPK cascade during JCPyV infection and the downstream signaling consequences, illuminating kinases as potential therapeutic targets for viral infection.

HTLV-1 as a Model for Virus and Host Coordinated Immunoediting.

Immunoediting is a process that occurs in cancer, whereby the immune system acts to initially repress, and subsequently promote the outgrowth of tumor cells through the stages of elimination, equilibrium, and escape. Here we present a model for a virus that causes cancer where immunoediting is coordinated through synergistic viral- and host-mediated events. We argue that the initial viral replication process of the Human T cell leukemia virus type I (HTLV-1), which causes adult T cell leukemia/lymphoma (ATL) in ~5% of individuals after decades of latency, harmonizes with the host immune system to create a population of cells destined for malignancy. Furthermore, we explore the possibility for HIV to fit into this model of immunoediting, and propose a non-malignant escape phase for HIV-infected cells that persist beyond equilibrium.

A framework for viral marketing replication and mutation

The value of successful viral marketing campaigns to marketing performance is widely acknowledged. As a result, a great deal of attention is paid to mechanisms that foster viral spread. However, due to the consumer-influenced nature of viral replication, it is also important to consider the malleability of such messages. Expanding the focus to include the potential for viral mutations is a critical step in understanding how viral messages evolve and in understanding which messages are most likely to spread and generate their desired impact. Further, there currently is no accepted model for the viral marketing replication process to guide research efforts. Therefore, this article defines viral message mutations, builds a framework for the viral replication process, and identifies susceptibility to mutation at various stages of that process. The proposed framework is then used to offer numerous avenues for future research.

Screening of Duck Tembusu Virus NS3 Interacting Host Proteins and Identification of Its Specific Interplay Domains.

NS3 protein is a member of the non-structural protein of duck Tembusu virus (DTMUV), which contains three domains, each of which has serine protease, nucleotide triphosphatase, and RNA helicase activities, respectively. It performs a variety of biological functions that are involved in the regulation of the viral life cycle and host immune response. Based on the yeast two-hybrid system, we successfully transformed pGBKT7-NS3 bait plasmid into Y2H Gold, tested it to prove that it has no self-activation and toxicity, and then hybridized it with the prey yeast strain of the duck embryo fibroblast cDNA library for screening. After high-stringency selection, positive alignment with the National Center for Biotechnology Information database revealed nine potential interactive proteins: MGST1, ERCC4, WIF1, WDR75, ACBD3, PRDX1, RPS7, ND5, and LDHA. The most interesting one (PRDX1) was selected to be verified with full-length NS3 protein and its three domains S7/DEXDc/HELICc using yeast regressive verification and GST Pull-Down assay. It denoted that PRDX1 does indeed interact with HELICc domains of NS3. NS3 is involved in the RNA uncoiling process of viral replication, which may cause mitochondrial overload to create oxidative stress (OS) during DTMUV attack. We deduced that the HELICc domain binding partner PRDX1, which regulates the p38/mitogen-activated protein kinase pathway (p38/MAPK) to avert OS, causing apoptosis, making it possible for viruses to escape host immune responses.

Role for Gag-CA Interdomain Linker in Primate Lentiviral Replication.

Gag proteins underlie retroviral replication by fulfilling numerous functional roles at various stages during viral life cycle. Out of the four mature proteins, Gag-capsid (CA) is a major component of viral particles, and has been most well studied biogenetically, biochemically and structurally. Gag-CA is composed of two structured domains, and also of a short stretch of disordered and flexible interdomain linker. While the two domains, namely, N-terminal and C-terminal domains (NTD and CTD), have been the central target for Gag research, the linker region connecting the two has been poorly studied. We recently have performed systemic mutational analyses on the Gag-CA linker region of HIV-1 by various experimental and in silico systems. In total, we have demonstrated that the linker region acts as a cis-modulator to optimize the Gag-related viral replication process. We also have noted, during the course of conducting the research project, that HIV-1 and SIVmac, belonging to distinct primate lentiviral lineages, share a similarly biologically active linker region with each other. In this brief article, we summarize and report the results obtained by mutational studies that are relevant to the functional significance of the interdomain linker of HIV/SIV Gag-CA. Based on this investigation, we discuss about the future directions of the research in this line.

The Peptide AmPep1 Derived from Amaranth Recognizes the Replication Hairpin of TYLCV Disturbing Its Replication Process in Host Plants.

Antiviral compounds targeting viral replicative processes have been studied as an alternative for the control of begomoviruses. Previously, we have reported that the peptide AmPep1 has strong affinity binding to the replication origin sequence of tomato yellow leaf curl virus (TYLCV). In this study, we describe the mechanism of action of this peptide as a novel alternative for control of plant-infecting DNA viruses. When AmPep1 was applied exogenously to tomato and Nicotiana benthamiana plants infected with TYLCV, a decrease in the synthesis of the two viral DNA strands (CS and VS) was observed, with a consequent delay in the development of disease progress in treated plants. The chemical mechanism of action of AmPep1 was deduced using Raman spectroscopy and molecular modeling showing the formation of chemical interactions such as H bonds and electrostatic interactions and the formation of π-π interactions between both biomolecules contributing to tampering with the viral replication.

Matrine inhibits IL-1\u03b2 secretion in primary porcine alveolar macrophages through the MyD88/NF-\u03baB pathway and NLRP3 inflammasome

Our previous studies demonstrated that matrine directly acts on the replication process of porcine reproductive and respiratory syndrome virus (PRRSV). Matrine inhibits viral replication and is also associated with the NF-κB signalling pathway. These results suggest that matrine has antiviral and anti-inflammatory effects. However, the specific anti-inflammatory mechanism of matrine is still unclear. In this study, we investigated the anti-IL-1β mechanism of matrine, as IL-1β is a major inflammatory cytokine, in porcine alveolar macrophages (PAMs) stimulated with 4 μg PRRSV 5′-untranslated region (UTR) RNA and 1 μg/mL LPS. After 5′UTR RNA and LPS co-stimulation of PAMs for 12 h, the expression of IL-1β, IL-6, IL-8 and TNF-α was significantly increased. The results also showed that co-stimulation induced the expression of MyD88, and activated the NF-κB signalling pathway and NLRP3 inflammasome. Furthermore, matrine treatment downregulated MyD88, NLRP3 and caspase-1 expression, inhibited ASC speck formation, suppressed IκBα phosphorylation, and interfered with the translocation of NF-κB from the cytoplasm to the nucleus. These results suggest that matrine plays an important role in PAMs co-stimulated with PRRSV 5′UTR RNA and LPS via its effect on NF-κB and the NLRP3 inflammasome. These findings lay the foundation for the exploration of the clinical application of matrine in PRRSV disease.

Conserved amino acids around the DIII-DI linker region of the Newcastle disease virus fusion protein are critical for protein folding and fusion activity.

Newcastle disease virus (NDV), an avian paramyxovirus, causes Newcastle disease (ND) which is a highly contagious and fatal viral disease affecting poultry and most species of birds. The fusion (F) protein of NDV mediates membrane fusion, which is essential to the processes of viral entry, replication, and dissemination. Although several domains of NDV F are known to have important effects on regulating the membrane fusion activity, the role of the region around domain III (DIII) and domain I (DI) still remains ill-defined. Site-directed mutagenesis was utilized to change the conserved amino acids at 269, 274, 277, 286, 287, 290, 295, and 297 to alanine in order to investigate the effects of these conserved amino acids around the DIII and DI linker region of the NDV F protein on fusion activity. It was found that five of these substitutions almost abolished fusion activity except for mutants I269A, Q286A, and N297A, which showed 57.1%, 161.1%, and 97.7% of the wt F level, respectively. Four (I274A, D277A, V287A, and P290A) of these five mutants likely result in interfering with folding or transporting of the molecule since these proteins were minimally expressed at the cell surface, formed aggregates, or not proteolytically cleaved. However, mutant L295A almost abolished fusion activity even with a similar level of cell surface expression. These data indicated that conserved amino acids around the DIII-DI linker region are critical for the folding of the F protein and have an important influence on fusion activity.

Host factors involved in HBV cccDNA formation and transcription

The genome of hepatitis B virus (HBV) is an incomplete double-stranded circular DNA, also known as relaxed circular (RC) DNA. RC-DNA needs to be converted to covalently closed circular (ccc) DNA during the replication process. cccDNA acts as the transcription template for viral RNAs. One of those viral RNAs, pgRNA, acts as the template in the reverse-transcription process, producing minus-DNA and plus-DNA. Subsequently, the two finally form RC-DNA and finish the viral genome replication process. The existence of cccDNA is the key obstacle for curing hepatitis type B and eliminating HBV antigen using medicines such as interferons or nucleos(t)ide analogues (NAs). Many host factors are involved in HBV cccDNA formation and transcription. We summarized some of these host factors and explained their related functions in HBV infection and replication, detailing the mechanisms of cccDNA formation and subsequent transcription. When HBV enters host cells, its surface protein L interacts with the receptor NTCP on the cell surface, allowing HBV to enter through endocytosis and leaving its envelope behind. Subsequently, the HBV genome, the RC-DNA, enters the cell nucleus, and leaves the empty nucleocapsid behind. The genome RC-DNA must convert into cccDNA to start the replication process. Many host factors participate in this process in various ways, including helping to remove special constructions, fixing the gaps and the single stand areas. These host factors include TDP1, TDP2, FEN1 and factors involved in the DNA damage response (DDR) system. This process also requires host DNA polymerase to extend DNA sequences, including POLK, POLH and POLL. cccDNA does not exist independently, but rather it exists in combination with many histones and nonhistones. The epigenetic modification of histone influences cccDNA transcription in various ways. For example, acetylization of H3 and H4 induces transcription and some acetylases such as CBP, p300, PCAF, HAT1, and KAT8 are involved. Interestingly, most of the host acetylases are recruited by two viral proteins, HBX and HBC, suggesting that these two virus proteins may help the virus to kidnap host factors for the benefit of the virus. In addition to epigenetic modification, cccDNA transcription also requires many cis-elements and host transcription factors. Many ubiquitous and liver-enriched host factors such as NF-1, sp-1, TBP, CREB, HNF3, HNF1, HNF4, C/EBP, PPARα, FXR, FTF, PGC-1αcombine with cis-elements in HBV genome, including promoter regions and enhancer regions. This combination induces or inhibits the cccDNA transcription process. In truth, HBV cccDNA formation and transcription processes require more host involvement than this article mentions. Many new factors and interactions as well as key regulators still waiting to be discovered. This article may supply suggestions for research into the detailed mechanism of the two processes of HBV cccDNA.

ZAP's stress granule localization is correlated with its antiviral activity and induced by virus replication.

Cellular antiviral programs encode molecules capable of targeting multiple steps in the virus lifecycle. Zinc-finger antiviral protein (ZAP) is a central and general regulator of antiviral activity that targets pathogen mRNA stability and translation. ZAP is diffusely cytoplasmic, but upon infection ZAP is targeted to particular cytoplasmic structures, termed stress granules (SGs). However, it remains unclear if ZAP’s antiviral activity correlates with SG localization, and what molecular cues are required to induce this localization event. Here, we use Sindbis virus (SINV) as a model infection and find that ZAP’s localization to SGs can be transient. Sometimes no apparent viral infection follows ZAP SG localization but ZAP SG localization always precedes accumulation of SINV non-structural protein, suggesting virus replication processes trigger SG formation and ZAP recruitment. Data from single-molecule RNA FISH corroborates this finding as the majority of cells with ZAP localization in SGs contain low levels of viral RNA. Furthermore, ZAP recruitment to SGs occurred in ZAP-expressing cells when co-cultured with cells replicating full-length SINV, but not when co-cultured with cells replicating a SINV replicon. ZAP recruitment to SGs is functionally important as a panel of alanine ZAP mutants indicate that the anti-SINV activity is correlated with ZAP’s ability to localize to SGs. As ZAP is a central component of the cellular antiviral programs, these data provide further evidence that SGs are an important cytoplasmic antiviral hub. These findings provide insight into how antiviral components are regulated upon virus infection to inhibit virus spread.

Encapsidation of viral RNA in Picornavirales: studies on cowpea mosaic virus demonstrate dependence on viral replication

To elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We show here that the remarkable specificity of packaging observed in CPMV is due to a functional linking between the two processes of viral replication and encapsidation. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication, which opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

Clinical and Haematological Disorders in Cats with Natural and Progressive Infection by Feline Leukemia Virus (FeLV)

Background: Feline leukemia virus (FeLV) is an important infectious agent in cats, responsible for great health damages and a large death amount. Among the most common clinical disorders caused by FeLV, severe hematological changes are related at the progressive infection, ordinarily presented as cytopenias. Regarding its oncogenic potential, lymphoma and leukemia are the most observed illness originated by the agent. Therefore, this study has the goal of describing and comparing clinical and hematological disorders in FeLV positive cats from a selected population.Materials, Methods & Results: Data of clinical evaluations of felines from a previously performed cross-sectional epidemiological study were compiled. This study obtained the prevalence of FeLV infected cats in Santa Catarina Plateau. Three groups were established from the original sample of 274 cats; Group 1 (control), FeLV negative and none clinical alterations (n = 80); Group 2, FeLV positive and none clinical changes (n = 9); Group 3, FeLV positive cats with clinical disorders (n = 29). Physical and hematological examination data were obtained from the medical records. The clinical changes at Group 3 were mucosal pallor (65.51% [19/29]), neurological disorders (20.69% [6/29]), lymphoma (20.69% [6/29]), coinfections (10.34% [3/29]) and leukemia (6.9% [2/29]). The most observed hematological disorders of Group 3 were anemia (65.51% [19/29]) and thrombocytopenia (62.7% [18/20]). At the complete blood count, the medium erythrocyte and eosinophil count, hemoglobin concentration and hematocrit were lower for Group 3 than the other two groups. The mean number of platelets count were higher for Group 1 than Group 2 and 3. The mean of the other variables remained between reference numbers. However, a descriptive analysis within the groups demonstrated that lymphopenia was observed in 34.48% (10/29) and neutropenia in 17.24% (5/29) at the Group 3. Discussion: The greater FeLV positive cats with clinical alterations number is a result of a sample obtained from a hospital population. In this scenario, cats are submitted to medical appointment in order to solve their clinical signs, not for purpose of infection screening. The frequency of non-neoplasic disease in FeLV positive cats was higher than neoplastic diseases. The mucosal pallor is the most important clinical change and it is an anemia consequence. A large number of clinical signs related in this study was non-specific. All the clinical changes are associated to FeLV infection, regardless the clinical syndrome observed. Neurological disorders frequency at this study was higher than other articles, being related to neoplastic lymphocytes in central nervous system at the resolved cases. Lymphoma and leukemia were the only neoplasms found in this FeLV study. Non-regenerative anemia was the main FeLV associated hematologic change. This disorder is originated by several mechanisms, such as the virus influence on bone marrow precursors and leukemic infiltration. Thrombocytopenia showed lower frequency than anemia and leukopenia, however, it was the second most common alteration in this study, which was observed in Group 2 and 3. The severe cytopenias related in Group 3 are probably due to the destruction or decrease of the production of these cells by the virus replication process. The clinical and hematological disorders found in this study, reveal the serious presentation and usually irreversible situation of this viral infection, originating important health harm to the affected cats.

The Role of Host Cytoskeleton in Flavivirus Infection.

The family of flaviviruses is one of the most medically important groups of emerging arthropod-borne viruses. Host cell cytoskeletons have been reported to have close contact with flaviviruses during virus entry, intracellular transport, replication, and egress process, although many detailed mechanisms are still unclear. This article provides a brief overview of the function of the most prominent flaviviruses-induced or -hijacked cytoskeletal structures including actin, microtubules and intermediate filaments, mainly focus on infection by dengue virus, Zika virus and West Nile virus. We suggest that virus interaction with host cytoskeleton to be an interesting area of future research.

Effects of high temperature on pandemic and seasonal human influenza viral replication and infection-induced damage in primary human tracheal epithelial cell cultures

High temperature reduces influenza viral replication; however, the treatment of fevers is thought to be necessary to improve patients' conditions. We examined the effects of high temperature on viral replication and infection-induced damage to human tracheal epithelial cells. Cell viability and dome formation were reduced, the number of detached cells was increased and lactate dehydrogenase (LDH) levels tended to be increased from 72 h to 120 h in uninfected cells cultured at 40 °C. Long-term (72 h and/or 120 h) exposure to high temperatures (39 °C and/or 40 °C) decreased RNA levels and/or viral titers of eight influenza virus strains. Cell viability and dome formation were reduced, and the number of detached cells and LDH levels were increased to a similar extent after infection with the A/H1N1 pdm 2009 virus at 37 °C and 40 °C. High temperature increased the endosomal pH, where the viral RNA enters the cytoplasm, in uninfected cells. High temperature reduced the production of IL-6, which mediate viral replication processes, and IL-1β and IL-8 in uninfected and infected cells. Based on these findings, high temperature may cause similar levels of airway cell damage after infection to cells exposed normal temperatures, although high temperature reduces viral replication by affecting the function of acidic endosomes and inhibiting IL-6-mediated processes.

Distinct role of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication

AimsTo investigate the role and underlying mechanism of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication. Main methodsThe mRNA relative expression of HBx and its DNA level were detected by real-time PCR. The relative levels of hepatitis B surface antigen (HBsAg) were measured by enzyme-linked immunosorbent assay (ELISA). HBx DNA level of HepG2 cells transfected with pcDNA3.1(+)-HBV1.3 plasmids were detected by Southern blot. Moreover, we determined autophagy through electron microscopy, confocal microscopy and Western blot. Key findingsRapamycin promoted autophagy and the X protein synthesis concomitantly with elevation in Akt phosphorylation and Beclin1 expression. Either Beclin1 or Akt depletion suppresses the Rapa-enhanced HBV replication, whereas mTOR silencing inhibited HBV replication concurring with a decreased in both S6K1 and 4E-BP1 phosphorylation. Unexpectedly, Akt inhibitor suppressed Rapa-dependent autophagic flux and increased the level of p62/SQSTM1. While S6K1 ablation impaired autophagy and decreased X protein expression, 4E-BP1 silencing slightly influenced autophagy and increased X protein level. SignificanceThe underlying mechanism of 4E-BP1 and S6K1, two main downstream effectors of mTOR, in mediating HBV replication and HBV-induced autophagy remains largely unknown. Here, we propose that Akt is required for both HBV replication and Rapa-induced autophagy, and 4E-BP1 and S6K1 play a distinct role in the virus replication and autophagic process.

Proteo-Transcriptomic Dynamics of Cellular Response to HIV-1 Infection

Throughout the HIV-1 replication cycle, complex host-pathogen interactions take place in the infected cell, leading to the production of new virions. The virus modulates the host cellular machinery in order to support its life cycle, while counteracting intracellular defense mechanisms. We investigated the dynamic host response to HIV-1 infection by systematically measuring transcriptomic, proteomic, and phosphoproteomic expression changes in infected and uninfected SupT1 CD4+ T cells at five time points of the viral replication process. By means of a Gaussian mixed-effects model implemented in the new R/Bioconductor package TMixClust, we clustered host genes based on their temporal expression patterns. We identified a proteo-transcriptomic gene expression signature of 388 host genes specific for HIV-1 replication. Comprehensive functional analyses of these genes confirmed the previously described roles of some of the genes and revealed novel key virus-host interactions affecting multiple molecular processes within the host cell, including signal transduction, metabolism, cell cycle, and immune system. The results of our analysis are accessible through a freely available, dedicated and user-friendly R/Shiny application, called PEACHi2.0. This resource constitutes a catalogue of dynamic host responses to HIV-1 infection that provides a basis for a more comprehensive understanding of virus-host interactions.