Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

Wilda, Maximiliano; López, Nora; Casabona, Juan Cruz; Franze-Fernández, Marı́a T.

doi:10.1128/jvi.01533-08

Cited by 35 publications

(39 citation statements)

References 33 publications

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“…1 E and F). Z has been identified as a binding partner to both host and viral proteins (15)(16)(17)(18)(31)(32)(33)(34). However, by establishing an in vitro model system of arenavirus RNA synthesis (Fig.…”

Section: Discussionmentioning

confidence: 99%

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Kranzusch

Whelan

2011

Proc. Natl. Acad. Sci. U.S.A.

104

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Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.transcription factor | Machupo virus | matrix protein | negative-strand RNA virus | gene regulation T ranscription initiation is a fundamental focal point of gene expression regulation in all orders of life. Modulation of RNA synthesis affords an important response to stress, alterations in growth conditions, and environmental cues. Although initiation of RNA synthesis is often indirectly controlled through gene promoters and accessory signaling elements, regulation also occurs through factors directly interacting with and altering the catalytic potential of the RNA synthesis machinery itself (1). Studies with phage T7 and the T7 lysozyme repressor system have provided an important paradigm for control of viral RNA transcription (2, 3), but these processes remain a mystery for many eukaryotic viruses and medically relevant pathogens. Gene expression regulation is particularly critical for negative-sense RNA viruses of the family Arenaviridae, which must respond to input from the host environment to maintain a noncytolytic infection in their rodent reservoir (4). In stark contrast to this persistent infection, arenavirus infection in humans can lead to severe hemorrhagic fever disease and many arenaviruses represent important emerging pathogens (5). Intriguingly, recent evidence indicates that the balance between persistent and acute viral infection can be altered by a single mutation within the viral polymerase (6). Mechanistic insight into how arenaviruses tune gene expression and control viral RNA synthesis is required to further our understanding of the response of arenavirus infection to various host and cellular environments.Arenaviruses are the simplest of all hemorrhagic fever viruses, with only four viral proteins. The viral genome consists of two segments of single-stranded negative-sense RNA, where each segment encodes two proteins in an ambisense orientation. The small segment encodes for the viral glycoprotein, essential for entr...

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Section: Discussionmentioning

confidence: 99%

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Kranzusch

Whelan

2011

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Most recently, budding by Z has been shown to be enhanced in the presence of viral NP (16). Z has also been shown to interact with NP and L (11,14,17,31,38), but the specific determinants in Z that mediate these viral protein interactions have yet to be fully elucidated.…”

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confidence: 99%

Conserved Residues in Lassa Fever Virus Z Protein Modulate Viral Infectivity at the Level of the Ribonucleoprotein

Capul

Torre

Buchmeier

2011

J Virol

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Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles.Arenaviruses are the causative agents of a number of human diseases, including lymphocytic choriomeningitis, Lassa fever, and Bolivian hemorrhagic fever. These viruses are carried asymptomatically in wild rodents and are transmitted through contact with contaminated excretions. Endemic infection in these rodents maintains a reservoir of virus that can reemerge in human populations as outbreaks (1,18,30) or as novel pathogenic species (6). No FDA-licensed vaccines exist for these viruses, and current antiarenaviral therapy is limited to the off-label use of the nucleoside analogue ribavirin, which has had mixed success in treating cases of arenaviral hemorrhagic fever (HF) disease, while being associated with significant side effects (5).The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is ubiquitous in wild rodents and can infect domesticated mice and hamsters. These infected hamsters have most recently been responsible for reported disease in humans (2, 34). Additionally, reports of transplant-associated LCMV disease and fatality highlight the clinical relevance of arenaviral infection in immunocompromised individuals (15). In addition, congenital transmission of LCMV has been implicated in a variety of clinical conditions that affect mainly the retina and brain (3, 4).Arenaviruses are enveloped viruses bearing a bi-segmented, negative-sense RNA genome. Each segment contains two open reading frames (ORFs) in the ambisense orientation that are separated by a noncoding intergenic region (IGR) and bounded at their 5Ј and 3Ј ends by untranslated regions (UTRs) that are critical for the control of viral R...

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“…Indeed, we previously demonstrated that the interaction of TCRV Z protein with two noncontiguous regions in the L polymerase inhibits viral RNA replication and transcription (23,52). Nevertheless, the precise molecular determinants in Z mediating its interaction with L have remained elusive.…”

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confidence: 99%

Molecular Determinants of Arenavirus Z Protein Homo-Oligomerization and L Polymerase Binding

Loureiro

Wilda

Macleod

et al. 2011

J Virol

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The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.

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Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

Cited by 35 publications

References 33 publications

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Conserved Residues in Lassa Fever Virus Z Protein Modulate Viral Infectivity at the Level of the Ribonucleoprotein

Molecular Determinants of Arenavirus Z Protein Homo-Oligomerization and L Polymerase Binding

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