Annual Review of Virology - Volume 4, 2017

My laboratory investigations have been driven by an abiding interest in understanding the consequences of genetic rearrangement in evolution and disease, and in using viruses to elucidate fundamental mechanisms in biology. Starting with bacteriophages and moving to the retroviruses, my use of the tools of genetics, molecular biology, biochemistry, and biophysics has spanned more than half a century—from the time when DNA structure was just discovered to the present day of big data and epigenetics. Both riding and contributing to the successive waves of technology, my laboratory has elucidated fundamental mechanisms in DNA replication, repair, and recombination. We have made substantial contributions in the area of retroviral oncogenesis, delineated mechanisms that control retroviral gene expression, and elucidated critical details of the structure and function of the retroviral enzymes—reverse transcriptase, protease, and integrase—and have had the satisfaction of knowing that the fundamental knowledge gained from these studies contributed important groundwork for the eventual development of antiviral drugs to treat AIDS. While pursuing laboratory research as a principal investigator, I have also been a science administrator—moving from laboratory head to department chair and, finally, to institute director. In addition, I have undertaken a number of community service, science-related “extracurricular” activities during this time. Filling all of these roles, while being a wife and mother, has required family love and support, creative management, and, above all, personal flexibility—with not too much long-term planning. I hope that this description of my journey, with various roles, obstacles, and successes, will be both interesting and informative, especially to young female scientists.

Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPR-Cas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action were elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field.

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Diverse prokaryotes produce gene transfer agents (GTAs), which are bacteriophage-like particles that exclusively package pieces of the producing cell's genome and transfer them to other cells. There are clear evolutionary connections between GTAs and phages, but GTAs have properties that lead us to suggest they are more than simply defective phages and instead provide a selective advantage for the producing organisms. The five types of currently known GTAs are genetically distinct, indicating multiple instances of convergent evolution. GTA production can be regulated by the producing organism and coordinated to coincide with development of the capability to receive DNA from GTAs. Recent discoveries of the genetic basis of GTA production in the bacterium Rhodobacter capsulatus and characterization of novel phages that possess homologs of this GTA's structural and regulatory genes have provided important new connections among these elements and highlight the tangled evolutionary relationships within the phageome.

Influenza A viruses are constantly changing. This change accounts for seasonal epidemics, infrequent pandemics, and zoonotic outbreaks. A major mechanism underlying the genetic diversification of influenza A virus is reassortment of intact gene segments between coinfecting viruses. This exchange is possible because of the segmented nature of the viral genome. Here, I first consider the constraints and drivers acting on influenza A virus reassortment, including the likelihood of coinfection at the host and cellular levels, mixing and assembly of heterologous gene segments within coinfected cells, and the fitness associated with reassortant genotypes. I then discuss the implications of reassortment for influenza A virus evolution, including its classically recognized role in the emergence of genetically “shifted” pandemic strains as well as its potential role as a catalyst of genetic drift.

Viruses must establish an intimate relationship with their hosts and vectors in order to infect, replicate, and disseminate; hence, viruses can be considered as symbionts with their hosts. Symbiotic relationships encompass different lifestyles, including antagonistic (or pathogenic, the most well-studied lifestyle for viruses), commensal (probably the most common lifestyle), and mutualistic (important beneficial partners). Symbiotic relationships can shape the evolution of the partners in a holobiont, and placing viruses in this context provides an important framework for understanding virus-host relationships and virus ecology. Although antagonistic relationships are thought to lead to coevolution, this is not always clear in virus-host interactions, and impacts on evolution may be complex. Commensalism implies a hitchhiking role for viruses—selfish elements just along for the ride. Mutualistic relationships have been described in detail in the past decade, and they reveal how important viruses are in considering host ecology. Ultimately, symbiosis can lead to symbiogenesis, or speciation through fusion, and the presence of large amounts of viral sequence in the genomes of everything from bacteria to humans, including some important functional genes, illustrates the significance of viral symbiogenesis in the evolution of all life on Earth.

Hemorrhagic fevers caused by viruses were identified in the late 1950s in South America. These viruses have existed in their hosts, the New World rodents, for millions of years. Their emergence as infectious agents in humans coincided with changes in the environment and farming practices that caused explosions in their host rodent populations. Zoonosis into humans likely occurs because the pathogenic New World arenaviruses use human transferrin receptor 1 to enter cells. The mortality rate after infection with these viruses is high, but the mechanism by which disease is induced is still not clear. Possibilities include direct effects of cellular infection or the induction of high levels of cytokines by infected sentinel cells of the immune system, leading to endothelia and thrombocyte dysfunction and neurological disease. Here we provide a review of the ecology and molecular and cellular biology of New World arenaviruses, as well as a discussion of the current animal models of infection. The development of animal models, coupled with an improved understanding of the infection pathway and host response, should lead to the discovery of new drugs for treating infections.

Circular single-stranded DNA viruses infect archaea, bacteria, and eukaryotic organisms. The relatively recent emergence of single-stranded DNA viruses, such as chicken anemia virus (CAV) and porcine circovirus 2 (PCV2), as serious pathogens of eukaryotes is due more to growing awareness than to the appearance of new pathogens or alteration of existing pathogens. In the case of the ubiquitous human circular single-stranded DNA virus family Anelloviridae, there is still no convincing direct causal relation to any specific disease. However, infections may play a role in autoimmunity by changing the homeostatic balance of proinflammatory cytokines and the human immune system, indirectly affecting the severity of diseases caused by other pathogens. Infections with CAV (family Anelloviridae, genus Gyrovirus) and PCV2 (family Circoviridae, genus Circovirus) are presented here because they are immunosuppressive and affect health in domesticated animals. CAV shares genomic organization, genomic orientation, and common features of major proteins with human anelloviruses, and PCV2 DNA may be present in human food and vaccines.

Chikungunya virus, first isolated in the 1950s, has since reemerged to cause several epidemics and millions of infections throughout the world. What was once blurred and confused with dengue virus in both diagnosis and name has since become one of the best-characterized arboviral diseases. In this review, we cover the history of this virus, its evolution into distinct genotypes and lineages, and, most notably, the convergent evolution observed in recent years. We highlight research that reveals to what extent convergent evolution, and its inherent predictability, may occur and what genetic or environmental factors may hinder it.

Viral abundance in soils can range from below detection limits in hot deserts to over 1 billion per gram in wetlands. Abundance appears to be strongly influenced by water availability and temperature, but a lack of informational standards creates difficulties for cross-study analysis. Soil viral diversity is severely underestimated and undersampled, although current measures of viral richness are higher for soils than for aquatic ecosystems. Both morphometric and metagenomic analyses have raised questions about the prevalence of nontailed, ssDNA viruses in soils. Soil is complex and critically important to terrestrial biodiversity and human civilization, but impacts of viral activities on soil ecosystem services are poorly understood. While information from aquatic systems and medical microbiology suggests the potential for viral influences on nutrient cycles, food web interactions, gene transfer, and other key processes in soils, very few empirical data are available. To understand the soil virome, much work remains.

Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses—a group of viruses that lack any lipid coating or envelope—play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

Viruses are completely dependent upon cellular machinery to support replication and have therefore developed strategies to co-opt cellular processes to optimize infection and counter host immune defenses. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode a relatively small number of genes. Viruses with limited genetic content often encode multifunctional proteins that function at multiple stages of the viral replication cycle. In this review, we discuss the functions of HIV-1 regulatory (Tat and Rev) and accessory (Vif, Vpr, Vpu, and Nef) proteins. Each of these proteins has a highly conserved primary activity; however, numerous additional activities have been attributed to these viral proteins. We explore the possibility that HIV-1 proteins leverage their multifunctional nature to alter host transcriptional networks to elicit a diverse set of cellular responses. Although these transcriptional effects appear to benefit the virus, it is not yet clear whether they are strongly selected for during viral evolution or are a ripple effect from the primary function. As our detailed knowledge of these viral proteins improves, we will undoubtedly uncover how the multifunctional nature of these HIV-1 regulatory and accessory proteins, and in particular their transcriptional functions, work to drive viral pathogenesis.

Although potent combination antiretroviral therapy can effectively block viral replication in the host, human immunodeficiency virus (HIV) persists due to the existence of latent but replication-competent proviruses residing primarily in a very small population of resting memory CD4⁺ T cells. Viral latency is established when the expression of the autoregulatory viral trans-activating factor Tat is reduced to subthreshold levels. The absence of Tat reduces HIV transcription and protein production to levels that make the host cell invisible to the immune system and refractory to antiretroviral treatment. Key host cell mechanisms that drive HIV into latency are sequestration of transcription initiation factors, establishment of epigenetic barriers inactivating the proviral promoter, and blockage of the assembly of the host elongation factor P-TEFb. This comprehensive understanding of the molecular control of HIV transcription is leading to the development of optimized combinatorial reactivation and immune surveillance strategies designed to purge the latent viral reservoir.

Recently, dozens of virus structures have been solved to resolutions between 2.5 and 5.0 Å by means of electron cryomicroscopy. With these structures we are now firmly within the “atomic age” of electron cryomicroscopy, as these studies can reveal atomic details of protein and nucleic acid topology and interactions between specific residues. This improvement in resolution has been the result of direct electron detectors and image processing advances. Although enforcing symmetry facilitates reaching near-atomic resolution with fewer particle images, it unfortunately obscures some biologically interesting components of a virus. New approaches on relaxing symmetry and exploring structure dynamics and heterogeneity of viral assemblies have revealed important insights into genome packaging, virion assembly, cell entry, and other stages of the viral life cycle. In the future, novel methods will be required to reveal yet-unknown structural conformations of viruses, relevant to their biological activities. Ultimately, these results hold the promise of answering many unresolved questions linking structural diversity of viruses to their biological functions.

The strong dependence of retroviruses, such as human immunodeficiency virus type 1 (HIV-1), on host cell factors is no more apparent than when the endosomal sorting complex required for transport (ESCRT) machinery is purposely disengaged. The resulting potent inhibition of retrovirus release underscores the importance of understanding fundamental structure-function relationships at the ESCRT–HIV-1 interface. Recent studies utilizing advanced imaging technologies have helped clarify these relationships, overcoming hurdles to provide a range of potential models for ESCRT-mediated virus abscission. Here, we discuss these models in the context of prior work detailing ESCRT machinery and the HIV-1 release process. To provide a template for further refinement, we propose a new working model for ESCRT-mediated HIV-1 release that reconciles disparate and seemingly conflicting studies.

Astroviruses are nonenveloped, positive-sense single-stranded RNA viruses that cause gastrointestinal illness. Although a leading cause of pediatric diarrhea, human astroviruses are among the least characterized enteric RNA viruses. However, by using in vitro methods and animal models to characterize virus-host interactions, researchers have discovered several important properties of astroviruses, including the ability of the astrovirus capsid to act as an enterotoxin, disrupting the gut epithelial barrier. Improved animal models are needed to study this phenomenon, along with the pathogenesis of astroviruses, particularly in those strains that can cause extraintestinal disease. Much like for other enteric viruses, the current dogma states that astroviruses infect in a species-specific manner; however, this assumption is being challenged by growing evidence that these viruses have potential to cross species barriers. This review summarizes these remarkable facets of astrovirus biology, highlighting critical steps toward increasing our understanding of this unique enteric pathogen.

In 1971, the first human polyomavirus was isolated from the brain of a patient who died from a rapidly progressing demyelinating disease known as progressive multifocal leukoencephalopathy. The virus was named JC virus after the initials of the patient. In that same year a second human polyomavirus was discovered in the urine of a kidney transplant patient and named BK virus. In the intervening years it became clear that both viruses were widespread in the human population but only rarely caused disease. The past decade has witnessed the discovery of eleven new human polyomaviruses, two of which cause unusual and rare cancers. We present an overview of the history of these viruses and the evolution of JC polyomavirus–induced progressive multifocal leukoencephalopathy over three different epochs. We review what is currently known about JC polyomavirus, what is suspected, and what remains to be done to understand the biology of how this mostly harmless endemic virus gives rise to lethal disease.

α, β, and θ defensins are effectors of the innate immune system with potent antibacterial, antiviral, and antifungal activity. Defensins have direct antiviral activity in cell culture, with varied mechanisms for individual viruses, although some common themes have emerged. In addition, defensins have potent immunomodulatory activity that can alter innate and adaptive immune responses to viral infection. In some cases, there is evidence for paradoxical escape from defensin neutralization or enhancement of viral infection. The direct and indirect activities of defensins have led to their development as therapeutics and vaccine components. The major area of investigation that continues to lag is the connection between the effects of defensins in cell culture models and viral pathogenesis in vivo. Model systems to study defensin biology, including more physiologic models designed to bridge this gap, are also discussed.