We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. IMPORTANCE Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life,Archaea. This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field.A rchaeal viruses constitute an integral part of the virosphere, and they are a ubiquitous feature of archaeal existence. Many Archaea are recognized as extremophiles, inhabiting extreme environments such as hot springs and solar salterns in high abundance (1, 2). Thus, viruses infecting extremophilic Archaea can be considered key players in the complex population dynamics in these environments. Through host infection, viruses can influence microbial diversity by introducing genetic variation, affect host cell physiology, and directly kill their hosts by cell lysis (3, 4). However, we have only a rudimentary understanding of archaeal virus-host interactions. Much of the limited knowledge that we do have comes from studying the virus-host interplay in Sulfolobales species. Of the about 100 isolated archaeal viruses, more than 30% infect hyperthermophilic Sulfolobales hosts (5-7). Among these viruses, distinct characteristics are found: unique bottle, droplet, and spindle shapes; extracellular virion development; and unique proteins with unknown functions (8). These distinctive characteristics are likely to influence the interplay with their hosts and give rise to unique life cycle traits. This has proven true for the rodshaped Sulfolobus virus ...
Sulfolobus mutants resistant to archaeal lytic virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) were isolated, and mutations were identified in two gene clusters, cluster sso3138 to sso3141 and cluster sso2386 and sso2387, encoding cell surface and type IV secretion proteins, respectively. The involvement of the mutations in the resistance was confirmed by genetic complementation. Blocking of virus entry into the mutants was demonstrated by the lack of early gene transcription, strongly supporting the idea of a role of the proteins in SIRV2 entry.T o date, relatively few archaeal viruses have been characterized, and most of those that have been characterized infect acidothermophilic members of the order Sulfolobales. Despite their limited number of around 50 species, they exhibit considerably greater morphological diversity than the more extensively characterized bacteriophages, about 95% of which show head-tail morphologies. Archaeal viruses, in contrast, exhibit fusiform shapes, often with one or two tails, bottle shapes, bearded-globular forms, and a wide variety of rod-like and filamentous morphotypes which often carry small terminal appendages (1-3). This morphological diversity suggests that the archaeal viruses may employ a variety of mechanisms to enter their hosts, but current insights into entry mechanisms are limited to an OppA transporter protein, Sso1273, possibly providing a receptor site for the Acidianus two-tailed virus (ATV) in Sulfolobus solfataricus P2 (4). And very recently, microscopic studies suggested that Sulfolobus islandicus rod-shaped virus 2 (SIRV2) enters the host cell by attaching and moving through a pilus-like filament; however, the nature of the structure and the identity of the involved proteins remain elusive (5).Sulfolobus solfataricus P2 is an acidothermophilic crenarchaeon that can host a wide range of archaeal viruses, many of which are propagated stably (1, 3, 6). Moreover, few of the viruses appear to induce cell lysis, possibly reflecting a need to minimize contact with the harsh hot acidic environment. However, recent studies have identified a few viruses that can enter a lytic phase, including the Sulfolobus turreted icosahedral virus (STIV), the two-tailed fusiform (ATV), and, more recently, the rudivirus SIRV2 (7-9).SIRV2 is classified in the family Rudiviridae together with other well-characterized viruses, including SIRV1 (10, 11), ARV1 (12) and SRV1, (13), all of which are rod shaped and lack an envelope, and their genomes consist of linear double-stranded DNA with covalently closed ends (10,14,15). In a recent microarray analysis of S. solfataricus infected with SIRV2, we demonstrated that the viral genes were activated at different times and that mainly stressresponse host genes and those implicated in vesicle formation were downregulated (16). The results also illustrated that SIRV2 infection at a multiplicity of infection (MOI) of 30 resulted in growth inhibition of S. solfataricus 5E6 (16). In the present experiment, the culture was infected at a lower...
Viral factories are compartmentalized centers for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by prototypical archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in the model archaeon Sulfolobus. This rod-shaped virus belongs to the viral family Rudiviridae, carrying linear double-stranded DNA (dsDNA) genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that of the four host DNA polymerases (DNA polymerase I [Dpo1] to Dpo4), only Dpo1 participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life. IMPORTANCEThe organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication center by a virus infecting members of the Archaea domain.KEYWORDS replication focus, archaeal virus, SIRV2, DNA polymerase T he compartmentalization of viral genome replication is a well-known feature of many eukaryotic viruses, where replication is confined to specific subcellular microenvironments termed viral factories, viroplasms, viral replication compartments, or viral replication centers. In general, viral factories function as a scaffold containing viral genomes and proteins involved in viral replication and assembly, and this is hypothesized to increase the efficiency of viral replication and confer protection from the cellular antiviral immune responses (1, 2).The formation of viral factories has been observed for DNA, double-stranded RNA (dsRNA), positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA viruses, and it has been extensively studied for nucleocytoplasmic large DNA viruses (e.g., poxviruses and asfarviruses) and positive-sense ssRNA viruses, including flaviviruses, coronaviruses, picornaviruses, and togaviruses (1). Although these viruses infect a wide range of hosts and their corresponding viral factories differ in their morphologies, components, and intracellular locations, their modes of formation share some similarities. First, formation of viral factories usually involves extensive organizational changes in the host cell membranes and/or cytoskeleton. Second, viral factories constitute a scaffold for the concentration of viral genomes and of viral and cellular proteins required for viral replication and assembly (1, 2).To date, the formation of viral factories has been studied mainly for eukaryotic viruses, although the organization of viral replication has been reported for bacteriophages 29, PRD1, SSP1, and 20...
Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional ‘brush-like’ structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.
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