Generation of Mutant Uukuniemi Viruses Lacking the Nonstructural Protein NSs by Reverse Genetics Indicates that NSs Is a Weak Interferon Antagonist

Abstract

Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the UnitedStates, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses. The Bunyaviridae are the largest grouping of RNA viruses, comprising Ͼ350 members. The family includes a wide range of enveloped, single-stranded RNA viruses with tripartite genomes of negative-sense or ambisense polarity. The Phlebovirus genus contains 70 viruses divided into two groups: the sandfly fever group, transmitted mainly by phlebotomines and mosquitoes, and the Uukuniemi-like virus group, transmitted by ticks. Uukuniemi virus (UUKV) itself was originally isolated in 1964 from an Ixodes ricinus tick in Finland (1). UUKV has served as a prototype tick-borne phlebovirus for many years and has aided in advances in bunyavirus structural and architectural determination (2-4), bunyavirus glycoprotein studies (5-9), and studies of bunyavirus entry into mammalian cells (10-12).Like other phleboviruses, the three UUKV genome segments, designated small (S), medium (M), and large (L), are found in the form of ribonucleoprotein (RNP) complexes in association with the nucleocapsid (N)...

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses

“…RVFV was the first phlebovirus in the Phenuiviridae family, for which a reverse genetics system was established to rescue infectious viral particles from cDNAs [36]. Since then, similar or derivative approaches have been adapted for some other phenuiviruses, including the tick-borne Uukuniemi virus (UUKV), and the severe fever with thrombocytopenia syndrome virus (SFTSV) [37][38][39]. We have here added a T7-based reverse genetics system enabling the modification of the TOSV viral genome.…”

Bunyaviruses: From Transmission by Arthropods to Virus Entry into the Mammalian Host First-Target Cells

“…Recent illustrations are Heartland virus (HRTV) in North America and severe fever with thrombocytopenia virus (SFTSV) in Asia, latter causing a fatality rate of up to 60% in some outbreaks [33][34][35][36][37]40]. In this context, there is a renewed interest in using UUKV to study these emerging viruses [42]. UUKV can be efficiently amplified in tick Ixodes ricinus cell lines [Mazelier M et al, Pers. Comm.…”

“…These systems were recently adapted to other bunyavirus members, including RVFV, UUKV, SFTSV and Schmallenberg virus [42,[103][104][105]. Reverse genetics has revolutionized negative-sense RNA virology.…”

In memoriam – Richard M. Elliott (1954–2015)

“…Bunyamwera virus rescue from cDNA is without doubt one of the most important contributions to bunyavirus molecular biology and the key achievement of Richard's career. In the subsequent years, Richard himself contributed major findings on viral virulence factors, gene regulation, promoter function, intracellular replication and particle assembly, and found ways to attenuate the viruses into safe and efficient vaccine candidates (Brennan et al, 2011b;Bridgen et al, 2001;Kohl et al, 2004Kohl et al, , 2006Lowen et al, 2005;Rezelj et al, 2015;Shi et al, 2007;TilstonLunel et al, 2015;van Knippenberg & Elliott, 2015). He published .130 papers, with many in the last few years and a considerable number in preparation at the time of his passing.…”

“…His group also pushed the flexibility of the viral genomes to the limit by creating a recombinant Bunyamwera virus that had an ambisense coding strategy more akin to the phleboviruses. Richard had many international and national collaborations, ensuring he was involved in all aspects of bunyavirology, even the seemingly impossible task of creating a Hantavirus reverse genetics system (Brennan et al, 2011a(Brennan et al, , b, 2014(Brennan et al, , 2015Elliott et al, 2013;MazelSanchez & Elliott, 2012;Rezelj et al, 2015;Tilston-Lunel et al, 2015;van Knippenberg & Elliott, 2015). Richard was a member of the International Committee on Taxonomy of Viruses Bunyavirus Study Group, an important service to the community.…”