A viral lncRNA tethers HSV-1 genomes at the nuclear periphery to establish viral latency.

IMPORTANCE: Herpes simplex virus 1 (HSV-1) establishes lifelong latency in neuronal cells. Following a stressor, the virus reactivates from latency, virus is shed at the periphery and recurrent disease can occur. During latency, the viral lncRNA termed the latency-associated transcript (LAT) is known to accumulate to high abundance. The LAT is known to impact many aspects of latency though the molecular events involved are not well understood. Here, we utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to identify the molecular-binding partners of the LAT during latency. We found that the LAT binds to both the cellular protein, TMEM43, and HSV-1 genomes in LUHMES cells. Additionally, we find that knockdown of TMEM43 prior to infection results in a decreased ability of HSV-1 to establish latency. This work highlights a potential mechanism for how the LAT facilitates the establishment of HSV-1 latency in human neurons.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.

Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread.

IMPORTANCE: Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.

HSV-1 exploits host heterochromatin for nuclear egress.

Herpes simplex virus (HSV-1) progeny form in the nucleus and exit to successfully infect other cells. Newly formed capsids navigate complex chromatin architecture to reach the inner nuclear membrane (INM) and egress. Here, we demonstrate by transmission electron microscopy (TEM) that HSV-1 capsids traverse heterochromatin associated with trimethylation on histone H3 lysine 27 (H3K27me3) and the histone variant macroH2A1. Through chromatin profiling during infection, we revealed global redistribution of these marks whereby massive host genomic regions bound by macroH2A1 and H3K27me3 correlate with decreased host transcription in active compartments. We found that the loss of these markers resulted in significantly lower viral titers but did not impact viral genome or protein accumulation. Strikingly, we discovered that loss of macroH2A1 or H3K27me3 resulted in nuclear trapping of capsids. Finally, by live-capsid tracking, we quantified this decreased capsid movement. Thus, our work demonstrates that HSV-1 takes advantage of the dynamic nature of host heterochromatin formation during infection for efficient nuclear egress.

Comprehensive Subcellular Localization of Swine Acute Diarrhea Syndrome Coronavirus Proteins.

Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV was first identified in diarrheal piglets in 2017. As a novel alphacoronavirus, SADS-CoV shares ~95% identity with bat alphacoronavirus HKU2. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Thus far, no effective antiviral drugs or vaccines are available to treat infections with SADS-CoV. Therefore, knowledge of the protein-coding gene set and a subcellular localization map of SADS-CoV proteins are fundamental first steps in this endeavor. Here, all SADS-CoV genes were cloned separately into Flag-tagged plasmids, and the subcellular localizations of viral proteins, with the exception of nsp11, were detected using confocal microscopy techniques. As a result, nsp1, nsp3-N, nsp4, nsp5, nsp7, nsp8, nsp9, nsp10, nsp14, and nsp15 were localized in the cytoplasm and nuclear spaces, and these viral proteins may perform specific functions in the nucleus. All structural and accessory proteins were mainly localized in the cytoplasm. NS7a and membrane protein M colocalized with the Golgi compartment, and they may regulate the assembly of SADS-CoV virions. Maturation of SADS-CoV may occur in the late endosomes, during which envelope protein E is involved in the assembly and release of the virus. In summary, the present study demonstrates for the first time the location of all the viral proteins of SADS-CoV. These fundamental studies of SADS-CoV will promote studies of basic virology of SADS-CoV and support preventive strategies for animals with infection of SADS-CoV. IMPORTANCE SADS-CoV is the first documented spillover of a bat coronavirus that causes severe diseases in domestic animals. Our study is an in-depth annotation of the newly discovered swine coronavirus SADS-CoV genome and viral protein expression. Systematic subcellular localization of SADS-CoV proteins can have dramatic significance in revealing viral protein biological functions in the subcellular locations. Furthermore, our study promote understanding the fundamental science behind the novel swine coronavirus to pave the way for treatments and cures.

Protein Myristoylation Plays a Role in the Nuclear Entry of the Parvovirus Minute Virus of Mice.

Being nonpathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses with great potential as anti-cancer agents. As these viruses replicate in the host cell nucleus, they must gain access to the nucleus during infection. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the nuclear envelope (NE) and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. In this study, we used MVM as a model to investigate the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays, mutagenesis, and pharmacological inhibition, we identified a functional myristoylation site at the sequence 78GGKVGH83 of the unique portion of the capsid protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this myristoylation site or with purified virions, we found tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying this cleavage exposes G (glycine) 78 at the N-terminus of VP1u for myristoylation. Subsequent experiments using inhibitors of myristoylation and cellular proteases with MVM-infected cells, or an imaging-based quantitative NE permeabilization assay, further indicate protein myristoylation and a chymotrypsin-like activity are essential for MVM to locally disrupt the NE during viral nuclear entry. We thus propose a model for the nuclear entry of MVM in which NE disruption is mediated by VP1u myristoylation after the intact capsid undergoes proteolytic processing to expose the required N-terminal G for myristoylation. IMPORTANCE Rodent parvoviruses (PVs), including minute virus of mice (MVM), have the ability to infect and kill cancer cells and thereby possess great potential in anti-cancer therapy. In fact, some of these viruses are currently being investigated in both preclinical studies and clinical trials to treat a wide variety of cancers. However, the detailed mechanism of how PVs enter the cell nucleus remains unknown. In this study, we for the first time demonstrated a chemical modification called "myristoylation" of a MVM protein plays an essential role in the nuclear entry of the virus. We also showed, in addition to protein myristoylation, a chymotrypsin-like activity, which may come from cellular proteasomes, is required for MVM to get myristoylated and enter the nucleus. These findings deepen our understanding on how MVM and other related PVs infect host cells and provide new insights for the development of PV-based anti-cancer therapies.

Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). vhs causes several changes to the infected cell environment that favor the translation of late (L) virus proteins: cellular mRNAs are degraded, immediate early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C terminus with GFP. When first expressed, vhs-GFP localized to juxtanuclear clusters, and later it colocalized and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, which is in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. These results revealed that the vhs-induced nuclear retention of IE and E transcripts was dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. IMPORTANCE Like many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here, we describe a virus expressing vhs tagged at its C terminus with a green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the early virus transcriptome in the nucleus to favor late protein translation, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus, therefore, has uncoupled the nuclear retention and degradation activities of vhs, providing a new understanding of vhs during infection.

Visualization of Marek's Disease Virus Genomes in Living Cells during Lytic Replication and Latency.

Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek's disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.

Proteomic Analysis of Nuclear Hepatitis B Virus Relaxed Circular DNA-Associated Proteins Identifies UV-Damaged DNA Binding Protein as a Host Factor Involved in Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the coprecipitated proteins by mass spectrometry. DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported short hairpin RNA (shRNA) screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex, and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of the DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Transcomplementation of wild-type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA binding, DDB1 binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified several host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNA interference (RNAi) screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay, we discovered a novel relationship between the UV-DDB complex and cccDNA formation, providing a proof of concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.

Identification of cellular proteins associated with human cytomegalovirus (HCMV) DNA replication suggests novel cellular and viral interactions.

Upon entry of Human cytomegalovirus (HCMV) into the host cell, the viral genome is transported to the nucleus where it serves as a template for transcription and genome replication. Production of new viral genomes is a coordinated effort between viral and cellular proteins. While the core replication proteins are encoded by the virus, additional cellular proteins support the process of genome synthesis. We used accelerated native isolation of proteins on nascent DNA (aniPOND) to study protein dynamics on nascent viral DNA during HCMV infection. Using this method, we identified specific viral and cellular proteins that are associated with nascent viral DNA. These included transcription factors, transcriptional regulators, DNA damage and repair factors, and chromatin remodeling complexes. The association of these identified proteins with viral DNA was confirmed by immunofluorescent imaging, chromatin-immunoprecipitation analyses, and shRNA knockdown experiments. These data provide evidence for the requirement of cellular factors involved in HCMV replication.

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.

Herpesviruses assimilate kinesin to produce motorized viral particles.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.

Disassembling the Nature of Capsid: Biochemical, Genetic, and Imaging Approaches to Assess HIV-1 Capsid Functions.

The human immunodeficiency virus type 1 (HIV-1) capsid and its disassembly, or capsid uncoating, has remained an active area of study over the past several decades. Our understanding of the HIV-1 capsid as solely a protective shell has since shifted with discoveries linking it to other complex replication events. The interplay of the HIV-1 capsid with reverse transcription, nuclear import, and integration has led to an expansion of knowledge of capsid functionality. Coincident with advances in microscopy, cell, and biochemistry assays, several models of capsid disassembly have been proposed, in which it occurs in either the cytoplasmic, nuclear envelope, or nuclear regions of the cell. Here, we discuss how the understanding of the HIV-1 capsid has evolved and the key methods that made these discoveries possible.

Nuclear Import of HIV-1.

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial-temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.

To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.

Nuclear restriction of HIV-1 infection by SUN1.

Overexpression of the human Sad-1-Unc-84 homology protein 2 (SUN2) blocks HIV-1 infection in a capsid-dependent manner. In agreement, we showed that overexpression of SUN1 (Sad1 and UNC-84a) also blocks HIV-1 infection in a capsid-dependent manner. SUN2 and the related protein SUN1 are transmembrane proteins located in the inner membrane of the nuclear envelope. The N-terminal domains of SUN1/2 localizes to the nucleoplasm while the C-terminal domains are localized in the nuclear lamina. Because the N-terminal domains of SUN1/2 are located in the nucleoplasm, we hypothesized that SUN1/2 might be interacting with the HIV-1 replication complex in the nucleus leading to HIV-1 inhibition. Our results demonstrated that SUN1/2 interacts with the HIV-1 capsid, and in agreement with our hypothesis, the use of N-terminal deletion mutants showed that SUN1/2 proteins bind to the viral capsid by using its N-terminal domain. SUN1/2 deletion mutants correlated restriction of HIV-1 with capsid binding. Interestingly, the ability of SUN1/2 to restrict HIV-1 also correlated with perinuclear localization of these proteins. In agreement with the notion that SUN proteins interact with the HIV-1 capsid in the nucleus, we found that restriction of HIV-1 by overexpression of SUN proteins do not block the entry of the HIV-1 core into the nucleus. Our results showed that HIV-1 restriction is mediated by the interaction of SUN1/2N-terminal domains with the HIV-1 core in the nuclear compartment.

RNA Sequencing of Medusavirus Suggests Remodeling of the Host Nuclear Environment at an Early Infection Stage.

Viruses of the phylum Nucleocytoviricota, or nucleo-cytoplasmic large DNA viruses (NCLDVs), undergo a cytoplasmic or nucleo-cytoplasmic cycle, the latter of which involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting NCLDVs. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate its infection strategy, we performed a time course RNA sequencing (RNA-seq) experiment. All viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and often (45%) encoded putative nucleus-localized proteins. These results suggest massive reshaping of the host nuclear environment by viral proteins at an early stage of infection. Genes in other expression clusters (clusters 2 to 5) were assigned to various functional categories. The virally encoded core histone genes were in cluster 3, whereas the viral linker histone H1 gene was in cluster 1, suggesting they have distinct roles during the course of the virus infection. The transcriptional profile of the host Acanthamoeba castellanii genes was greatly altered postinfection. Several encystment-related host genes showed increased representation levels at 48 h postinfection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. IMPORTANCE Medusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family "Medusaviridae" in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA sequencing (RNA-seq) analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intranuclear environment during infection. The transcriptional profile of amoeba genes was greatly altered postinfection.

Bromodeoxyuridine Labelling to Determine Viral DNA Localization in Fluorescence and Electron Microscopy: The Case of Adenovirus.

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.