Search

Viruses exploit key host cell factors to accomplish each individual stage of the viral replication cycle. To understand viral pathogenesis and speed the development of new antiviral strategies, high-resolution visualization of virus-host interactions is needed to define where and when these events occur within cells. Here, we review state-of-the-art live cell imaging techniques for tracking individual stages of viral life cycles, focusing predominantly on retroviruses and especially human immunodeficiency virus type 1, which is most extensively studied. We describe how visible viruses can be engineered for live cell imaging and how nonmodified viruses can, in some instances, be tracked and studied indirectly using cell biosensor systems. We summarize the ways in which live cell imaging has been used to dissect the retroviral life cycle. Finally, we discuss select challenges for the future including the need for better labeling strategies, increased resolution, and multivariate systems that will allow for the study of full viral replication cycles.

Members of the family Retroviridae are important animal and human pathogens. Being obligate parasites, their replication involves a series of steps during which the virus hijacks the cellular machinery. Additionally, many of the steps of retrovirus replication are unique among viruses, including reverse transcription, integration, and specific packaging of their genomic RNA (gRNA) as a dimer. Progress in retrovirology has helped identify several molecular mechanisms involved in each of these steps, but many are still unknown or remain controversial. This review summarizes our present understanding of the molecular mechanisms involved in various stages of retrovirus replication. Furthermore, it provides a comprehensive analysis of our current understanding of how different retroviruses package their gRNA into the assembling virions. RNA packaging in retroviruses holds a special interest because of the uniqueness of packaging a dimeric genome. Dimerization and packaging are highly regulated and interlinked events, critical for the virus to decide whether its unspliced RNA will be packaged as a "genome" or translated into proteins. Finally, some of the outstanding areas of exploration in the field of RNA packaging are highlighted, such as the role of epitranscriptomics, heterogeneity of transcript start sites, and the necessity of functional polyA sequences. An in-depth knowledge of mechanisms that interplay between viral and cellular factors during virus replication is critical in understanding not only the virus life cycle, but also its pathogenesis, and development of new antiretroviral compounds, vaccines, as well as retroviral-based vectors for human gene therapy., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bone morphogenetic protein (BMP) is a kind of classical multi-functional growth factor that plays a vital role in the formation and maintenance of bone, cartilage, muscle, blood vessels, and the regulation of adipogenesis and thermogenesis. However, understanding of the role of BMPs in antiviral immunity is still limited. Here we demonstrate that Bmp8a is a newly-identified positive regulator for antiviral immune responses. The bmp8a -/- zebrafish, when infected with viruses, show reduced antiviral immunity and increased viral load and mortality. We also show for the first time that Bmp8a interacts with Alk6a, which promotes the phosphorylation of Tbk1 and Irf3 through p38 MAPK pathway, and induces the production of type I interferons (IFNs) in response to viral infection. Our study uncovers a previously unrecognized role of Bmp8a in regulation of antiviral immune responses and provides a target for controlling viral infection.

One of the most important steps in any viral lifecycle is the production of progeny virions. For retroviruses as well as other viruses, this step is a highly organized process that occurs with exquisite spatial and temporal specificity on the cellular plasma membrane. To facilitate this process, retroviruses encode short peptide motifs, or L domains, that hijack host factors to ensure completion of this critical step. One such cellular machinery targeted by viruses is known as the Endosomal Sorting Complex Required for Transport (ESCRTs). Typically responsible for vesicular trafficking within the cell, ESCRTs are co-opted by the retroviral Gag polyprotein to assist in viral particle assembly and release of infectious virions. This review in the Viruses Special Issue "The 11th International Retroviral Nucleocapsid and Assembly Symposium", details recent findings that shed light on the molecular details of how ESCRTs and the ESCRT adaptor protein ALIX, facilitate retroviral dissemination at sites of viral assembly.

Small ubiquitin-like modifier (SUMO)ylation is a crucial post-translational modification that controls functions of a wide collection of proteins and biological processes. Hence, given its pleiotropic role, viruses have developed many approaches to usurp SUMO conjugation to exploit the cellular host environment for their own benefit. Consistently, cancer cells also frequently impact on SUMO to force cellular transformation, underlining the importance of SUMO in health and diseases. Therefore, after a brief introduction to the multistep SUMOylation pathway, in this review we will focus our attention on several examples of strategies adopted by oncogenic viruses to hijack SUMOylation in order to promote infection, persistence and malignant transformation of host cells.

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission., (Copyright © 2019 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Through this activity, they are implicated in almost every cellular process investigated to date. Hence, it is not surprising that miRNAs play diverse roles in regulation of viral infections and antiviral responses. Diverse families of DNA and RNA viruses have been shown to take advantage of cellular miRNAs or produce virally encoded miRNAs that alter host or viral gene expression. MiRNA-mediated changes in gene expression have been demonstrated to modulate viral replication, antiviral immune responses, viral latency, and pathogenesis. Interestingly, viruses mediate both canonical and non-canonical interactions with miRNAs to downregulate specific targets or to promote viral genome stability, translation, and/or RNA accumulation. In this review, we focus on recent findings elucidating several key mechanisms employed by diverse virus families, with a focus on miRNAs at the host⁻virus interface during herpesvirus, polyomavirus, retroviruses, pestivirus, and hepacivirus infections.

Retroviruses are obligate intracellular parasites of eukaryotic cells. After reverse transcription, the viral DNA contained in the preintegration complex is delivered to the nucleus of the host cell, where it integrates. Before reaching the nucleus, the incoming particle and the preintegration complex must travel throughout the cytoplasm. Likewise, the newly synthesized viral proteins and viral particles must transit the cytoplasm during exit. The cytoplasm is a crowded environment, and simple diffusion is difficult. Therefore, viruses have evolved to utilize the cellular mechanisms of movement through the cytoplasm, where microtubules are the roads, and the ATP-dependent motors dynein and kinesin are the vehicles for retrograde and anterograde trafficking. This review will focus on how different retroviruses (Mazon-Pfizer monkey virus, prototype foamy virus, bovine immunodeficiency virus, human immunodeficiency virus type 1, and murine leukemia virus) have subjugated the microtubule-associated motor proteins for viral replication. Although there have been advances in our understanding of how retroviruses move along microtubules, the strategies are different among them. Thus, a better understanding of the mechanisms used by each retrovirus to functionally subvert microtubule motor proteins will provide important clues in the design of new antiretroviral drugs that can specifically disrupt intracellular viral trafficking., (© 2017 John Wiley & Sons Ltd.)

Viral epitranscriptomics is a newly emerging field that has identified unique roles for RNA modifications in modulating life cycles of RNA viruses. Despite the observation of a handful of modified viral RNAs five decades ago, very little was known about how these modifications regulate viral life cycles, until recently. Here we review the pro- and anti-viral effects of methyl-6-adenosine in distinct viral life cycles, the role of 2' O-methyl modifications in RNA stability and innate immune sensing, and functions of adenosine to inosine modifications in retroviral life cycles. With roles for over 100 modifications in RNA still unknown, this is a rapidly emerging field that is destined to suggest novel antiviral therapies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The observation that the Runx genes act as targets for transcriptional activation by retroviral insertion identified a new family of dominant oncogenes. However, it is now clear that Runx genes are 'conditional' oncogenes whose over-expression is growth inhibitory unless accompanied by another event such as concomitant over-expression of MYC or loss of p53 function. Remarkably, while the oncogenic activities of either MYC or RUNX over-expression are suppressed while p53 is intact, the combination of both neutralises p53 tumour suppression in vivo by as yet unknown mechanisms. Moreover, there is emerging evidence that endogenous, basal RUNX activity is important to maintain the viability and proliferation of MYC-driven lymphoma cells. There is also growing evidence that the human RUNX genes play a similar conditional oncogenic role and are selected for over-expression in end-stage cancers of multiple types. Paradoxically, reduced RUNX activity can also predispose to cell immortalisation and transformation, particularly by mutant Ras. These apparently conflicting observations may be reconciled in a stage-specific model of RUNX involvement in cancer. A question that has yet to be fully addressed is the extent to which the three Runx genes are functionally redundant in cancer promotion and suppression.

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang et al., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription., (Copyright © 2016. Published by Elsevier Inc.)

A fascinating aspect of retroviruses is their tendency to nonrandomly incorporate host cell RNAs into virions. In addition to the specific tRNAs that prime reverse transcription, all examined retroviruses selectively package multiple host cell noncoding RNAs (ncRNAs). Many of these ncRNAs appear to be encapsidated shortly after synthesis, before assembling with their normal protein partners. Remarkably, although some packaged ncRNAs, such as pre-tRNAs and the spliceosomal U6 small nuclear RNA (snRNA), were believed to reside exclusively within mammalian nuclei, it was demonstrated recently that the model retrovirus murine leukemia virus (MLV) packages these ncRNAs from a novel pathway in which unneeded nascent ncRNAs are exported to the cytoplasm for degradation. The finding that retroviruses package forms of ncRNAs that are rare in cells suggests several hypotheses for how these RNAs could assist retrovirus assembly and infectivity. Moreover, recent experiments in several laboratories have identified additional ways in which cellular ncRNAs may contribute to the retrovirus life cycle. This review focuses on the ncRNAs that are packaged by retroviruses and the ways in which both encapsidated ncRNAs and other cellular ncRNAs may contribute to retrovirus replication., (Copyright © 2016 Eckwahl et al.)

Since the introduction of retroviral vector technology, permanent genetic marking of cells has considerably contributed to the understanding of different physiological and disease processes in vivo. Recent marking strategies aim to elucidate the contribution of cells on the clonal level, and the advent of fluorescent proteins has opened new avenues for the in vivo analysis of gene-marked cells. Gene-modified cells are easily identifiable (e.g., via the introduced fluorescent protein) within whole organ structures, allowing one to measure the contribution of transduced cells to malignant outgrowth. In our laboratory, we use the tetracycline-inducible system to study oncogene cooperation in metastatic progression. We use bicistronic retroviruses expressing the tetracycline transactivator (tTA) and the candidate gene (MIT-gene) or the tTA alone (MIT-Rx) to infect primary mammary cells from mice harboring tetracycline-inducible transgenes. This allows for constitutive expression of the candidate gene and tTA-dependent expression of the inducible oncogene. We also use MIG-based vectors, which allow for constitutive expression of the candidate gene and a green fluorescent protein. Here we describe how to produce retroviral particles carrying both MIT- and MIG-based vectors. Because of the fragility of the retroviral envelope, we do not attempt to concentrate the virus, and we directly use packaging cell media to infect primary epithelial cells (either normal or tumor). Infected cells can be transplanted into recipient mice to investigate metastatic colonization., (© 2016 Cold Spring Harbor Laboratory Press.)

Unlabelled: TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins., Importance: Restriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5α inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5α itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5α is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5α does not require autophagic machinery. These data indicate that the effector functions of TRIM5α can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype., (© 2015 Wiley Periodicals, Inc.)

Hepatic progenitor cells isolated from fetal liver can be transplanted into recipient mice to reconstitute an organ system. Retroviral infection can be used to introduce putative oncogenes or short-hairpin RNAs (shRNAs) targeting putative tumor suppressors into the cells; reconstituted mice then develop livers that are chimeric for cells bearing a specific alteration. This approach has the advantage of examining tumorigenesis on a largely wild-type background (if only a subset of cells are infected), a situation that more accurately parallels the human situation. Additionally, tumor development occurs within the appropriate native microenvironment. Here, we describe the isolation of hepatic progenitor cells, as well as the reconstitution and tumor monitoring of recipient mice., (© 2015 Cold Spring Harbor Laboratory Press.)

Stem cell transplantation is well established in humans for the treatment of hematopoietic disease, including hematopoietic malignancies. Similar direct transplant procedures can readily be performed in mice; these procedures can be paired with retroviral infection to introduce exogenous genes or to silence endogenous genes in a subset of cells in the murine hematopoietic system. The resulting mice are chimeric for cells bearing a specific alteration. This approach has the advantage of examining tumorigenesis on a largely wild-type background (if only a subset of cells are infected), a situation that more accurately parallels the human situation. Additionally, tumor development occurs within the appropriate native microenvironment. Here, we describe the isolation and retroviral infection of hematopoietic stem cells (HSCs), as well as the reconstitution and monitoring of tumor formation in lethally irradiated recipient mice. This protocol requires a source of long-term HSCs; these can include either stimulated adult bone marrow or fetal liver—the site of primitive hematopoiesis., (© 2015 Cold Spring Harbor Laboratory Press.)

Many important aspects of human retroviral infections cannot be fully evaluated using only in vitro systems or unmodified animal models. An alternative approach involves the use of humanized mice, which consist of immunodeficient mice that have been transplanted with human cells and/or tissues. Certain humanized mouse models can support robust infection with human retroviruses including different strains of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). These models have provided wide-ranging insights into retroviral biology, including detailed information on primary infection, in vivo replication and pathogenesis, latent/persistent reservoir formation, and novel therapeutic interventions. Here we describe the humanized mouse models that are most commonly utilized to study retroviral infections, and outline some of the important discoveries that these models have produced during several decades of intensive research., (Copyright © 2015 Elsevier Inc. All rights reserved.)