Structural maturation of rubella virus in the Golgi complex

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

Section: Discussionsupporting

confidence: 52%

Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

Shi

Kohl

Léonard

et al. 2006

“…There are many examples of viral maturation events that occur within the Golgi complex and secretory vesicles (32,44,49,51). Most recently, structural changes in the West Nile virus capsid have been determined by cryo-electron microscopy as the virus particles travel through the low-pH environment of the TGN (72).…”

Section: Vol 81 2007 Dynamic Interactions Of Ul16 With Hsv Capsids mentioning

confidence: 99%

Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus

Meckes

Wills

2007

138

The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines.Herpesviruses are assembled with more than 40 different virally encoded proteins that comprise three morphologically distinct structures: the icosahedral nucleocapsid containing the viral DNA; the lipid envelope containing virus-encoded glycoproteins; and the tegument, the assortment of proteins between the nucleocapsid and the envelope. The specific mechanisms by which these various viral proteins come together to drive the assembly and budding processes are poorly understood.The addition of tegument proteins to capsids during assembly is widely viewed as an ordered process of adding "layers" of protein (38). In this model, some tegument is added in the nucleus, more in the cytoplasm, and the rest at the trans-Golgi network (TGN) where maturation budding occurs (39). The data presented in this report challenge this view and suggest that the process is more dynamic than previously recognized. Specifically, the UL16 protein of herpes simplex virus (HSV) may be the first example of a tegument protein that associates with and releases from capsids during egress through low-pH compartments of the cell.The UL16 protein is conserved throughout the herpesvirus family (41,45,69). As measured by immunofluorescence, this protein has been found to b...

“…Some of these complex viruses use the Golgi apparatus as a key element of the viral factory (41,43,44). The reasons for this preference are not clear since it adds a potential difficulty in terms of efficient transport of large structures, such as viral particles, along the Golgi stack.…”

mentioning

confidence: 99%

“…We have been studying the structure and assembly pathways of several complex enveloped viruses to understand how viral factors interact with cellular elements to generate infectious virions (11,34,40,41,43,44). Bunyaviruses in particular are an adequate structural model to analyze how the viral factory is built and how Golgi factors participate in viral assembly and maturation.…”

mentioning

confidence: 99%

Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus

Novoa

Calderita

Cabezas

et al. 2005

Self Cite

Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a transGolgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-␤-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.