Viruses of the Thermotogota; the vehicles on the highways of gene sharing

Viruses are drivers of microbial ecology and evolution controlling populations and disseminating DNA laterally among the cells they infect. Phylogenetic and comparative genomic analyses of Thermotogota bacteria have shown high levels of lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely source of such DNA transfers is viruses, however, to date only three temperate viruses infecting Marinitoga bacteria have been characterized in this phylum. Here we use a bioinformatic approach to identify an additional 17 proviruses integrated into genomes of eight Thermotogota genera including Marinitoga. We also induce viral particle production from one of the newly identified proviruses. The proviruses fall into two groups based on sequence similarities, gene synteny and virus classification tools. Group-1 shows similar genome structure to the three previously identified Marinitoga viruses while Group-2 are distantly related to these viruses and have different genome organization. Both groups show close connections to Firmicutes in genomic- and phylogenetic analyses, and the Group-2 viruses show evidence of very recent transfer between these lineages and are likely capable of infecting cells from both phyla. We suggest that viruses are responsible for a large portion of the laterally transferred DNA between these distantly related lineages.

Herpesviral Latency—Common Themes

Latency establishment is the hallmark feature of herpesviruses, a group of viruses, of which nine are known to infect humans. They have co-evolved alongside their hosts, and mastered manipulation of cellular pathways and tweaking various processes to their advantage. As a result, they are very well adapted to persistence. The members of the three subfamilies belonging to the family Herpesviridae differ with regard to cell tropism, target cells for the latent reservoir, and characteristics of the infection. The mechanisms governing the latent state also seem quite different. Our knowledge about latency is most complete for the gammaherpesviruses due to previously missing adequate latency models for the alpha and beta-herpesviruses. Nevertheless, with advances in cell biology and the availability of appropriate cell-culture and animal models, the common features of the latency in the different subfamilies began to emerge. Three criteria have been set forth to define latency and differentiate it from persistent or abortive infection: 1) persistence of the viral genome, 2) limited viral gene expression with no viral particle production, and 3) the ability to reactivate to a lytic cycle. This review discusses these criteria for each of the subfamilies and highlights the common strategies adopted by herpesviruses to establish latency.

Nipah Virus-Like Particle Egress Is Modulated by Cytoskeletal and Vesicular Trafficking Pathways: a Validated Particle Proteomics Analysis

ABSTRACT Classified as a biosafety level 4 (BSL4) select agent, Nipah virus (NiV) is a deadly henipavirus in the Paramyxoviridae family, with a nearly 75% mortality rate in humans, underscoring its global and animal health importance. Elucidating the process of viral particle production in host cells is imperative both for targeted drug design and viral particle-based vaccine development. However, little is understood concerning the functions of cellular machinery in paramyxoviral and henipaviral assembly and budding. Recent studies showed evidence for the involvement of multiple NiV proteins in viral particle formation, in contrast to the mechanisms understood for several paramyxoviruses as being reliant on the matrix (M) protein alone. Further, the levels and purposes of cellular factor incorporation into viral particles are largely unexplored for the paramyxoviruses. To better understand the involvement of cellular machinery and the major structural viral fusion (F), attachment (G), and matrix (M) proteins, we performed proteomics analyses on virus-like particles (VLPs) produced from several combinations of these NiV proteins. Our findings indicate that NiV VLPs incorporate vesicular trafficking and actin cytoskeletal factors. The involvement of these biological processes was validated by experiments indicating that the perturbation of key factors in these cellular processes substantially modulated viral particle formation. These effects were most impacted for NiV-F-modulated viral particle formation either autonomously or in combination with other NiV proteins, indicating that NiV-F budding relies heavily on these cellular processes. These findings indicate a significant involvement of the NiV fusion protein, vesicular trafficking, and actin cytoskeletal processes in efficient viral particle formation. IMPORTANCE Nipah virus is a zoonotic biosafety level 4 agent with high mortality rates in humans. The genus to which Nipah virus belongs, Henipavirus, includes five officially recognized pathogens; however, over 20 species have been identified in multiple continents within the last several years. As there are still no vaccines or treatments for NiV infection, elucidating its process of viral particle production is imperative both for targeted drug design as well as for particle-based vaccine development. Developments in high-throughput technologies make proteomic analysis of isolated viral particles a highly insightful approach to understanding the life cycle of pathogens such as Nipah virus.

Gymnotic Delivery of LNA Mixmers Targeting Viral SREs Induces HIV-1 mRNA Degradation

Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers “nakedly”, without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.

THE FUNCTIONAL E1–E2 HETERODIMER HCV GLYCOPROTEINS

Introduction: The two HCV envelope glycoproteins E1 and E2 are released fromHCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembraneproteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobicanchor. Methods and pathways: After their synthesis, HCV glycoproteins E1 and E2 associateas a non covalent heterodimer. The transmembrane domains of HCV envelope glycoproteinsplay a major role in E1–E2 heterodimer assembly and subcellular localization. The envelopeglycoprotein complex E1–E2 has been proposed to be essential for HCV entry. Results andconclusions: However, for a long time, HCV entry studies have been limited by the lack of arobust cell culture system for HCV replication and viral particle production. Recently, a modelmimicking the entry process of HCV lifecycle has been developed by pseudo typing retroviralparticles with native HCV envelope glycoproteins, allowing the characterization of functionalE1–E2 envelope glycoproteins., we review our understanding to date on the assembly of thefunctional HCV glycoprotein heterodimer.

The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from WT cells. These results suggest that this Y45F mutation in NS4A causes secretion of low density Core particles devoid of genomic HCV RNA. These results corroborate previous findings showing that mutation of the first hydrophobic region of E1 also causes secretion of Core complexes lacking RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.Author SummaryRNA viruses, which encompass both established and emerging pathogens, pose significant public health challenges. Viruses in the familyFlavivirdae, including Dengue virus, Zika virus and hepatitis C virus (HCV), continue to cause morbidity and mortality worldwide. One HCV protein, NS4A, has known functions in several steps of the viral lifecycle, however, how it contributes to viral particle production is not understood. Here, we investigated the role of one region of NS4A, the C-terminal acidic domain, in regulating the viral lifecycle. We found that some of the amino acids within this domain are important for viral envelopment to make infectious particles, specifically through interaction with the E1 glycoprotein. NS4A interacts with the first hydrophobic domain of E1. Disruption of this interaction prevents the production of infectious virus particles and instead results in release of low density Core protein complexes that lack HCV RNA into the cellular supernatant. Overall, our results reveal that NS4A is important for late stages of the HCV lifecycle and suggest that the interaction between NS4A and E1 may regulate the incorporation of viral RNA into the virion for the formation of infectious HCV particles.

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

ABSTRACTINI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel ofSAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic intranscomplementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-stategagRNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown ofINI1reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events.IMPORTANCESignificant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, includinggagRNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.