BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ-binding motif to the cytoplasm.

The carboxy-terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work, we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies nor by the PDZ domain-containing protein-binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

Liquid-liquid phase separation drives herpesvirus assembly in the cytoplasm.

Liquid-liquid phase separation (LLPS) has emerged as a fundamental mechanism to compartmentalize biomolecules into membraneless organelles. In this issue, Zhou et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202201088), report that MHV-68 ORF52 undergoes LLPS to form cytoplasmic virion assembly compartments, regulating the spatiotemporal compartmentalization of viral components.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

Herpesvirus Nuclear Egress across the Outer Nuclear Membrane.

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress.

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates.

HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/ß-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/ß-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/ß-catenin signaling components and on transcriptional regulation of WNT/ß-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

SAFA initiates innate immunity against cytoplasmic RNA virus SFTSV infection.

Nuclear scaffold attachment factor A (SAFA) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS with a high fatality rate of up to 30%. It remains elusive whether and how cytoplasmic SFTSV can be sensed by the RNA sensor SAFA. Here, we demonstrated that SAFA was able to detect SFTSV infection and mediate antiviral interferon and inflammatory responses. Transcription and expression levels of SAFA were strikingly upregulated under SFTSV infection. SAFA was retained in the cytoplasm by interaction with SFTSV nucleocapsid protein (NP). Importantly, SFTSV genomic RNA was recognized by cytoplasmic SAFA, which recruited and promoted activation of the STING-TBK1 signaling axis against SFTSV infection. Of note, the nuclear localization signal (NLS) domain of SAFA was important for interaction with SFTSV NP and recognition of SFTSV RNA in the cytoplasm. In conclusion, our study reveals a novel antiviral mechanism in which SAFA functions as a novel cytoplasmic RNA sensor that directly recognizes RNA virus SFTSV and mediates an antiviral response.

New Cytoplasmic Virus-Like Elements (VLEs) in the Yeast Debaryomyces hansenii.

Yeasts can have additional genetic information in the form of cytoplasmic linear dsDNA molecules called virus-like elements (VLEs). Some of them encode killer toxins. The aim of this work was to investigate the prevalence of such elements in D. hansenii killer yeast deposited in culture collections as well as in strains freshly isolated from blue cheeses. Possible benefits to the host from harboring such VLEs were analyzed. VLEs occurred frequently among fresh D. hansenii isolates (15/60 strains), as opposed to strains obtained from culture collections (0/75 strains). Eight new different systems were identified: four composed of two elements and four of three elements. Full sequences of three new VLE systems obtained by NGS revealed extremely high conservation among the largest molecules in these systems except for one ORF, probably encoding a protein resembling immunity determinant to killer toxins of VLE origin in other yeast species. ORFs that could be potentially involved in killer activity due to similarity to genes encoding proteins with domains of chitin-binding/digesting and deoxyribonuclease NucA/NucB activity, could be distinguished in smaller molecules. However, the discovered VLEs were not involved in the biocontrol of Yarrowia lipolytica and Penicillium roqueforti present in blue cheeses.

The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking.

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

Viroplasms: Assembly and Functions of Rotavirus Replication Factories.

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals.

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus-host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus-host interactions, we studied the life cycle of the enveloped, ∼2-µm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus-host interactions in Archaea.

Proteome Analysis in PAM Cells Reveals That African Swine Fever Virus Can Regulate the Level of Intracellular Polyamines to Facilitate Its Own Replication through ARG1.

In 2018, African swine fever broke out in China, and the death rate after infection was close to 100%. There is no effective and safe vaccine in the world. In order to better characterize and understand the virus-host-cell interaction, quantitative proteomics was performed on porcine alveolar macrophages (PAM) infected with ASFV through tandem mass spectrometry (TMT) technology, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The proteome difference between the simulated group and the ASFV-infected group was found at 24 h. A total of 4218 proteins were identified, including 306 up-regulated differentially expressed proteins and 238 down-regulated differentially expressed proteins. Western blot analysis confirmed changes in the expression level of the selected protein. Pathway analysis is used to reveal the regulation of protein and interaction pathways after ASFV infection. Functional network and pathway analysis can provide an insight into the complexity and dynamics of virus-host cell interactions. Further study combined with proteomics data found that ARG1 has a very important effect on ASFV replication. It should be noted that the host metabolic pathway of ARG1-polyamine is important for virus replication, revealing that the virus may facilitate its own replication by regulating the level of small molecules in the host cell.

Role of Tunneling Nanotubes in Viral Infection, Neurodegenerative Disease, and Cancer.

The network of tunneling nanotubes (TNTs) represents the filamentous (F)-actin rich tubular structure which is connected to the cytoplasm of the adjacent and or distant cells to mediate efficient cell-to-cell communication. They are long cytoplasmic bridges with an extraordinary ability to perform diverse array of function ranging from maintaining cellular physiology and cell survival to promoting immune surveillance. Ironically, TNTs are now widely documented to promote the spread of various pathogens including viruses either during early or late phase of their lifecycle. In addition, TNTs have also been associated with multiple pathologies in a complex multicellular environment. While the recent work from multiple laboratories has elucidated the role of TNTs in cellular communication and maintenance of homeostasis, this review focuses on their exploitation by the diverse group of viruses such as retroviruses, herpesviruses, influenza A, human metapneumovirus and SARS CoV-2 to promote viral entry, virus trafficking and cell-to-cell spread. The later process may aggravate disease severity and the associated complications due to widespread dissemination of the viruses to multiple organ system as observed in current coronavirus disease 2019 (COVID-19) patients. In addition, the TNT-mediated intracellular spread can be protective to the viruses from the circulating immune surveillance and possible neutralization activity present in the extracellular matrix. This review further highlights the relevance of TNTs in ocular and cardiac tissues including neurodegenerative diseases, chemotherapeutic resistance, and cancer pathogenesis. Taken together, we suggest that effective therapies should consider precise targeting of TNTs in several diseases including virus infections.

Plant viral proteins and fibrillarin: the link to complete the infective cycle.

The interaction between viruses with the nucleolus is already a well-defined field of study in plant virology. This interaction is not restricted to those viruses that replicate in the nucleus, in fact, RNA viruses that replicate exclusively in the cytoplasm express proteins that localize in the nucleolus. Some positive single stranded RNA viruses from animals and plants have been reported to interact with the main nucleolar protein, Fibrillarin. Among nucleolar proteins, Fibrillarin is an essential protein that has been conserved in sequence and function throughout evolution. Fibrillarin is a methyltransferase protein with more than 100 methylation sites in the pre-ribosomal RNA, involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. Recently, it was found that AtFib2 shows a ribonuclease activity. In plant viruses, Fibrillarin is involved in long-distance movement and cell-to-cell movement, being two highly different processes. The mechanism that Fibrillarin performs is still unknown. However, and despite belonging to very different viral families, the majority comply with the following. (1) They are positive single stranded RNA viruses; (2) encode different types of viral proteins that partially localize in the nucleolus; (3) interacts with Fibrillarin exporting it to the cytoplasm; (4) the viral protein-Fibrillarin interaction forms an RNP complex with the viral RNA and; (5) Fibrillarin depletion affects the infective cycle of the virus. Here we review the relationship of those plant viruses with Fibrillarin interaction, with special focus on the molecular processes of the virus to sequester Fibrillarin to complete its infective cycle.

Sugarcane mosaic virus remodels multiple intracellular organelles to form genomic RNA replication sites.

Positive-stranded RNA viruses usually remodel the host endomembrane system to form virus-induced intracellular vesicles for replication during infections. The genus Potyvirus of the family Potyviridae represents the largest number of positive single-stranded RNA viruses, and its members cause great damage to crop production worldwide. Although potyviruses have a wide host range, each potyvirus infects a relatively limited number of host species. Phylogenesis and host range analysis can divide potyviruses into monocot-infecting and dicot-infecting groups, suggesting that they differ in their infection mechanisms, probably during replication. Comprehensive studies on the model dicot-infecting turnip mosaic virus have shown that the 6K2-induced replication vesicles are derived from the endoplasmic reticulum (ER) and subsequently target chloroplasts for viral genome replication. However, the replication site of monocot-infecting potyviruses is unknown. In this study, we show that the precursor 6K2-VPg-Pro polyproteins of dicot-infecting potyviruses and monocot-infecting potyviruses cluster phylogenetically in two separate groups. With a typical gramineae-infecting potyvirus-sugarcane mosaic virus (SCMV)-we found that replicative double-stranded RNA (dsRNA) forms aggregates in the cytoplasm but does not associate with chloroplasts. SCMV 6K2-VPg-Pro-induced vesicles colocalize with replicative dsRNA. Moreover, SCMV 6K2-VPg-Pro-induced structures target multiple intracellular organelles, including the ER, Golgi apparatus, mitochondria, and peroxisomes, and have no evident association with chloroplasts.

Annulate lamellae and intracellular pathogens.

Annulate lamellae (AL) have been observed many times over the years on electron micrographs of rapidly dividing cells, but little is known about these unusual organelles consisting of stacked sheets of endoplasmic reticulum-derived membranes with nuclear pore complexes (NPCs). Evidence is growing for a role of AL in viral infection. AL have been observed early in the life cycles of the hepatitis C virus (HCV) and, more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggesting a specific induction of mechanisms potentially useful to these pathogens. Like other positive-strand RNA viruses, these viruses induce host cells membranes rearrangements. The NPCs of AL could potentially mediate exchanges between these partially sealed compartments and the cytoplasm. AL may also be involved in regulating Ca2+ homeostasis or cell cycle control. They were recently observed in cells infected with Theileria annulata, an intracellular protozoan parasite inducing cell proliferation. Further studies are required to clarify their role in intracellular pathogen/host-cell interactions.

HBV Core Protein Is in Flux between Cytoplasmic, Nuclear, and Nucleolar Compartments.

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly.

Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication.

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.