The Equine Herpesvirus 1 UL34 Gene Product Is Involved in an Early Step in Virus Egress and Can Be Efficiently Replaced by a UL34-GFP Fusion Protein

126

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.Cytomegaloviruses define the beta subgroup of the Herpesviridae, which are important animal and human pathogens. Human cytomegalovirus (HCMV) is detected with a high prevalence in the human population, and it causes severe and fatal disease in immunocompromised individuals. Murine cytomegalovirus (MCMV) infection serves as an animal model system. CMVs harbor large double-stranded DNA genomes of about 230 kbp (7, 32). The recent cloning of the HCMV and MCMV genomes as infectious bacterial artificial chromosomes (BACs) (4,15,24,27,45,50) rendered these genomes accessible to advanced genetic manipulation (for a review, see reference 46).Infectious herpesvirus particles are formed during lytic virus replication as a result of a complex maturation and egress process (for a review, see reference 28). This multistep process starts in the nucleus with the packaging of viral genomes into capsids. The final envelopment takes place in the trans-Golgi network. Each maturation step is controlled by particular multiprotein assemblies, which are formed by complex interactions between viral and cellular proteins. Efforts to reconstitute the first morphogenesis stage, the capsid assembly, have been successful for alphaherpesviruses (43). In the mature virion, some higher-order protein complexes were studied by crystallography and electron microscopy (for a review, see reference 9). Yet, the logistics of the dynamic transitory stages (e.g., capsid egress from the nucleus) are not accessible for high-resolution analysis using static physical methods. Artificial reconstitution of these intermediate stages would require regulated coexpression of a number of viral proteins in the context of an appropriate host cell environment. Thus, detailed structure-function analyses of protein machines, which are associated with nuclear egress of virus capsids, need the context of virus replication. Gene products involved in herpesvirus morphogenesis are often essential for viral growth. The genetic analysis of these genes is restricted to either the isolated expression of the protein in cell ...

Section: Resultsmentioning

confidence: 99%

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication

Bubeck

Wagner

Ruzsics

et al. 2004

126

“…In the case of HSV-2, UL34 was detected on mature virions (40), while both the presence and absence of HSV-1 UL34 in the virion have been documented (36,50). EHV-1 UL34 was not detectable in extracellular virions (30), and in PrV it was visualized on intracellular virions but not on the mature extracellular virions (17). The latter behavior was considered further evidence in favor of the envelopment-de-envelopment-reenvelopment process of alphaherpesviruses.…”

Section: Discussionmentioning

confidence: 96%

“…With many similarities and a few differences, accumulating evidence indicates that these proteins and their homologs play similar roles in nuclear egress of both alpha-and betaherpesviruses (9,17,23,30,33,34,36,37,40,48,50). The physical interaction between the two proteins appears to be important to facilitate virion envelopment at the inner nuclear membrane, a process which probably also involves nuclear lamina disruption, to allow nucleocapsids to gain access at the interior face of the nuclear envelope (28, 39).…”

mentioning

confidence: 99%

Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina

Gonnella

Farina

Santarelli

et al. 2005

105

135

. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.Two conserved herpesvirus proteins, designated UL34 and UL31, of herpes simplex virus (HSV) and pseudorabies virus (PrV) are involved in the early steps of viral maturation at the nuclear envelope (reviewed in reference 26). With many similarities and a few differences, accumulating evidence indicates that these proteins and their homologs play similar roles in nuclear egress of both alpha-and betaherpesviruses (9,17,23,30,33,34,36,37,40,48,50). The physical interaction between the two proteins appears to be important to facilitate virion envelopment at the inner nuclear membrane, a process which probably also involves nuclear lamina disruption, to allow nucleocapsids to gain access at the interior face of the nuclear envelope (28, 39).We initially identified and characterized the prod...

“…The NEC contains homologs of the herpes simplex virus (HSV) UL31 and UL34 proteins (called pUL31 and pUL34), and these are critical for nuclear egress in all herpesviruses tested (9,15,24,25,33). pUL31 and pUL34 homologs interact with each other, and formation of a pUL31/pUL34 complex is required for proper targeting of the complex to the nuclear envelope (NE) (10,16,30,31,36,37,41).…”

mentioning

confidence: 99%

Intragenic and Extragenic Suppression of a Mutation in Herpes Simplex Virus 1 UL34 That Affects both Nuclear Envelope Targeting and Membrane Budding

Roller

Haugo

Kopping

2011

Late in infection herpesviruses move DNA-filled capsids from the nucleus to the cytoplasm by enveloping DNA-containing capsids at the inner nuclear membrane (INM) and deenveloping them at the outer nuclear membrane. This process requires two conserved herpesvirus proteins, pUL31 and pUL34. Interaction between pUL34 and pUL31 is essential for targeting both proteins to the nuclear envelope (NE), and sequences that mediate the targeting interaction have been mapped in both proteins. Here, we show that a mutation in the INM-targeting domain of pUL34 fails to support production of infectious virus or plaque formation. The mutation results in multiple defects, including impaired interaction between pUL34 and pUL31, poor NE targeting of pUL34, and misregulated, capsid-independent budding of the NE. The mutant defects in virus production, plaque formation, and pUL31 interaction can be suppressed by other mutations in the INMtargeting domain of pUL31 and by additional mutations in the pUL34 coding sequence.Efficient nuclear egress of herpesviruses requires formation of a nuclear envelopment complex (NEC) consisting of viral and cellular proteins (24,26,34). The NEC contains homologs of the herpes simplex virus (HSV) UL31 and UL34 proteins (called pUL31 and pUL34), and these are critical for nuclear egress in all herpesviruses tested (9,15,24,25,33). pUL31 and pUL34 homologs interact with each other, and formation of a pUL31/pUL34 complex is required for proper targeting of the complex to the nuclear envelope (NE) (10,16,30,31,36,37,41). The interactions that underlie complex formation are therefore critical for assembly and egress of all herpesviruses.Despite the conservation of a pUL31-pUL34 interaction, it is not clear that the structural basis for that interaction is completely conserved. The sequence of pUL31 and its homologs can be divided into four conserved regions (CRs) (Fig. 1B) (37). The most N-terminal of these regions (CR1) has been shown to mediate interaction with pUL34 homologs in examples from all herpesvirus subfamilies (19, 37). The situation with pUL34 homologs is less clear. For HSV-1 pUL34, the sequence that interacts with pUL31 and that is required for nuclear envelope targeting was mapped by deletion and domain swapping to amino acids (aa) 137 to 181 (18). This corresponds to the third of three CRs in the pUL34 sequence (Fig. 1A). Consistent with this, a construct containing CR1, CR2, and most of CR3 (aa 1 to 161) of pseudorabies virus (PRV) pUL34 was sufficient to interact with pUL31 in a yeast two-hybrid assay (10). In mouse cytomegalovirus (MCMV), on the other hand, use of small insertions and point mutations implicated a different region of the UL34 homolog, M50, in binding to the UL31 homolog, M53 (4,19,28). The interaction region is located in a highly conserved stretch of residues at the N terminus of M50 CR2. Whether the differences between MCMV M50 and HSV pUL34 reflect a very different structural basis for interaction is not yet clear.At the NE, pUL34 and pUL31 mediate subsequent steps ...