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

Abstract

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 ...

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication

“…2 C and D) all decreased affinity by at least fivefold. This segment of UL50 and M50, particularly E56 and Y57, and the corresponding segment of their pseudorabies virus and Kaposi's sarcoma herpes virus homologs have previously been shown to be important for interaction with the other NEC subunit in less quantitative coimmunoprecipitation and colocalization assays (16,19,32,33). We also found that several substitutions that alter residues between positions 121 and 129 of M50 and 125 and 139 of UL50 (at the top of the groove and within the cavity in Fig.…”

“…These residues are found in a loop just after helix-α2. This loop is quite distal to the core interaction surface, and other insertions nearby (between K36 and N37 and between C43 and D44) permit M53 binding (16). Interestingly, substitutions of HSV-1 UL34 residues corresponding to the β1-strand of M50, which is in the vicinity of this loop, also result in a dominant negative phenotype without affecting subunit interactions as assessed by colocalization (38,39).…”

“…MCMV M50 is a 316-aa protein with a predicted C-terminal transmembrane region (11) and an N-terminal one-half that is highly conserved among herpesvirus homologs (Figs. S1A and S2) (16,19). The NMR structure of this N-terminal conserved core (M50; residues 1-168) was determined using ∼1,400 NOE-derived distance constraints, of which ∼350 were long-distance constraints.…”

“…In HCMV, the NEC is comprised of UL50, which is an INM protein, and UL53, which is a nucleoplasmic protein that is brought to the INM by its interaction with UL50. These two proteins and their murine CMV (MCMV) homologues, M50 and M53, are essential for replication and nuclear egress (8,(14)(15)(16)(17) of their respective viruses. Although a process similar to herpesvirus nuclear egress was recently described for movement of ribonucleoprotein particles during Drosophila myogenesis (18), no host cell homolog of the NEC that would serve as mediator of this mechanism has yet been identified.…”

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

“…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.…”