bioRxiv preprint doi: https://doi.org/10.1101/2021.03.08.434518; this version posted March 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 High Transcriptional Activity and Diverse Functional Repertoires of Hundreds of 2 Giant Viruses in a Coastal Marine System 3 4 Anh D Ha, Mohammad Moniruzzaman, Frank O Aylward* 5 6 Department of Biological Sciences, Virginia Tech 7 926 West Campus Drive. Blacksburg, VA, 24061 8 *Correspondence: [email protected] 9 10 Running Title: Transcriptional Activity of Marine Giant Viruses 11 Keywords: Giant Viruses, NCLDV, Nucleocytoviricota, Viral Diversity, Kinesin, Myosin 12 13 Abstract 14 Viruses belonging to the Nucleocytoviricota phylum are globally distributed and include 15 members with notably large genomes with complex functional repertoires. Recent studies have 16 shown that these viruses are particularly diverse and abundant in marine systems, but the 17 magnitude of actively replicating Nucleocytoviricota present in ocean habitats remains unclear. 18 In this study, we compiled a curated database of 2,431 Nucleocytoviricota genomes and used it 19 to examine the gene expression of these viruses in a 2.5-day metatranscriptomic time-series from 20 surface waters of the California Current. We identified 145 viral genomes with high levels of 21 gene expression, including 90 Mimiviridae and 53 Phycodnaviridae. In addition to recovering 22 high expression of core genes involved in information processing that are commonly expressed 23 during viral infection, we also identified transcripts of diverse viral metabolic genes from 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.08.434518; this version posted March 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 pathways such as glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting that 25 virus-mediated reprogramming of central carbon metabolism is common in oceanic surface 26 waters. Surprisingly, we also identified viral transcripts with homology to actin, myosin, and 27 kinesin domains, suggesting that viruses may use them to manipulate host cytoskeletal dynamics 28 during infection. We performed phylogenetic analysis on the virus-encoded myosin and kinesin 29 proteins, which demonstrated that most belong to deep-branching viral clades, but that others 30 appear to have been acquired from eukaryotes more recently. Our results highlight a remarkable 31 diversity of active Nucleocytoviricota in a coastal marine system and underscore the complex 32 functional repertoires expressed by these viruses during infection. 33 34 Importance 35 The discovery of giant viruses has transformed our understanding of viral complexity. Although 36 viruses have traditionally been viewed as filterable infectious agents that lack metabolism, giant 37 viruses can reach sizes rivalling cellular lineages and possess genomes encoding central 38 metabolic processes. Recent studies have shown that giant viruses are widespread in aquatic 39 systems, but the activity of these viruses and the extent to which they reprogram host physiology 40 in situ remains unclear. Here we show that numerous giant viruses consistently express central 41 metabolic enzymes in a coastal marine system, including components of glycolysis, the TCA 42 cycle, and other pathways involved in nutrient homeostasis. Moreover, we found expression of 43 several viral-encoded actin, myosin, and kinesin genes, indicating viral manipulation of host 44 cytoskeleton during infection. Our study reveals a high activity of giant viruses in a coastal 45 marine system and indicates they are a diverse and underappreciated component of microbial 46 diversity in the ocean. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.08.434518; this version posted March 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 48 Introduction 49 Large dsDNA viruses of the family Nucleocytoviricota, commonly referred to as “giant viruses”, 50 are a diverse group of double-stranded DNA viruses with virion sizes reaching 1.5 µm, 51 comparable to the sizes of many cellular lineages (1–4). These viruses are known to infect a 52 broad range of eukaryotic hosts; members of the Poxviridae and Iridoviridae families infect 53 numerous metazoans, the Mimiviridae and Phycodnaviridae are known to infect a wide range of 54 algae and other protists, and the Asfarviridae infect a mixture of metazoan and protist hosts (5– 55 7). Members of the Nucleocytoviricota typically harbor exceptionally large genomes that are 56 often > 300 kbp in length, and in some cases as large as 2.5 Mbp (8). Numerous studies have 57 noted the unusually complex genomic repertoires of viruses in this group that include many 58 genes typically found only in cellular lineages, such as those involved in the TCA cycle (9, 10), 59 glycolysis (10), amino acid metabolism (11), light sensing (12, 13), sphingolipid biosynthesis 60 (14, 15), eukaryotic cytoskeleton (16), and fermentation (16). These complex metabolic 61 repertoires are thought to play a role in the manipulation of host physiology during infection, in 62 effect transforming healthy cells into reprogrammed “virocells” that more efficiently produce 63 viral progeny (17, 18). 64 The genomic complexity of Nucleocytoviricota together with reports that they are abundant in 65 global marine environments has raised questions regarding the extent to which they manipulate 66 the physiology of their hosts and thereby influence global biogeochemical cycles (19). Cultivated 67 representatives of these viruses are known to infect a broad array of ecologically-important 68 eukaryotic algae, including dinoflagellates, haptophytes, brown algae, and chlorophytes, among 69 others (20–23), suggesting they could play important roles in shaping the activity and 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.08.434518; this version posted March 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 composition of the microbial eukaryotic community. Even so, our understanding of the 71 distribution and activity of Nucleocytoviricota in marine systems has lagged behind that of other 72 viral groups owing to their large size, which precludes their presence in the small size fractions 73 typically analyzed in viral diversity studies (24). Some early studies were prescient in suggesting 74 that these viruses, and in particular those within the families Mimiviridae and Phycodnaviridae, 75 are a diverse and underappreciated component of viral diversity in the ocean (25–28). More 76 recent work has confirmed this view and revealed that a diverse range of Nucleocytoviricota 77 inhabit the global ocean and likely contribute to key processes such as algal bloom termination 78 and carbon export (10, 29–34). Some metatranscriptomic studies have also begun to note the 79 presence of Nucleocytoviricota transcripts in marine samples, confirming their activity (35–37). 80 Although these studies have vastly expanded our knowledge of the diversity and host range of 81 Nucleocytoviricota in the ocean, the activity of these viruses and the extent to which they use 82 cellular metabolic genes they encode during infection remains unclear. 83 In this study, we constructed a database of 2,436 annotated Nucleocytoviricota genomes for the 84 purpose of evaluating the gene expression landscape of these viruses. We then leveraged a 85 previously published metatranscriptome time-series from surface waters of the California 86 Current (37) to assess the daily activity of Nucleocytoviricota in this environment. We show that 87 hundreds of these viruses, primarily of the Mimiviridae and Phycodnaviridae families, were 88 consistently active during this period and frequently expressed genes involved in central carbon 89 metabolism, light harvesting, oxidative stress reduction, and lipid metabolism. Unexpectedly, we 90 also found expression of several viral-encoded cytoskeleton genes, including those that encode 91 the motor proteins myosin and kinesin, and we performed a phylogenetic analysis demonstrating 92 that Nucleocytoviricota commonly encode deep-branching enzymes in these protein families. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.08.434518; this version posted March 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 Our findings highlight the surprisingly high activity of Nucleocytoviricota in marine systems and 94 suggest they are an underappreciated component of viral diversity in the ocean. 95 96 97 Results and Discussion 98 We examined a metatranscriptomic dataset of 16 timepoints sampled over a 60-hour period that 99 were obtained from microbial communities previously reported for the California current system 100 (37). This dataset is ideal for examining the activity of marine Nucleocytoviricota because it 101 targeted the >5 µm size fraction, which is enriched in many of the eukaryotic plankton that these 102 viruses are known to infect. Altogether, we identified 145 Nucleocytoviricota genomes with 103 metatranscriptomic