Similar to other positive-strand RNA viruses, rhinovirus, the causative agent of the common cold, replicates on a web of cytoplasmic membranes, orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of the replication membranes and complexes are poorly understood. We show that rhinovirus replication depends on host factors driving phosphatidylinositol 4-phosphate (PI4P)-cholesterol counter-currents at viral replication membranes. Depending on the virus type, replication required phosphatidylinositol 4-kinase class 3beta (PI4K3b), cholesteryl-esterase hormone-sensitive lipase (HSL) or oxysterol-binding protein (OSBP)-like 1, 2, 5, 9, or 11 associated with lipid droplets, endosomes, or Golgi. Replication invariably required OSBP1, which shuttles cholesterol and PI4P between ER and Golgi at membrane contact sites. Infection also required ER-associated PI4P phosphatase Sac1 and phosphatidylinositol (PI) transfer protein beta (PITPb) shunting PI between ER-Golgi. These data support a PI4P-cholesterol counter-flux model for rhinovirus replication.

Two conserved herpes simplex virus 1 proteins, UL31 and UL34, form a complex at the inner nuclear membrane which governs primary envelopment and nuclear egress of the herpesvirus nucleocapsids. In mouse cytomegalovirus, a member of the betaherpesvirus subfamily, the homologous proteins M53/p38 and M50/p35 form the nuclear egress complex (NEC). Since the interaction of these proteins is essential for functionality, the definition of the mutual binding sites is a prerequisite for further analysis. Lytic replication of the herpesviruses gives rise to the production of infectious virus particles as a result of an assembly process that starts in the nucleus of the infected cell, where the viral genomes are packaged into the nucleocapsid. Nucleocapsid formation is followed by a complex process of nucleocapsid transitions through cellular membranes. First, the nucleocapsid buds from the inner nuclear membrane (INM) into the space between the INM and the outer nuclear membrane (ONM). This process is termed primary envelopment. By primary envelope fusion with the ONM, the nucleocapsids reach the cytoplasm. A secondary envelopment occurs at a not yet clearly defined compartment of the cellular exocytotic pathway. Finally, enveloped virus particles are released into the extracellular space (17,18).Virus maturation events are controlled by multiprotein assemblies which require the interaction of a number of viral and cellular proteins. Studies of alphaherpesviruses have shown that two conserved viral proteins, the prototypic UL31 and UL34 gene products, play a major role during primary envelopment (7,8,10,(25)(26)(27). In the betaherpesvirus mouse cytomegalovirus (MCMV), the products of the M53 and M50 genes (3, 7, 10, 19) have this function, and in the gammaherpesvirus Epstein-Barr virus, the BFRF1 and BFLF2 gene products (7, 10, 12) have this function. UL34 and related proteins are type II membrane proteins which, upon isolated expression, circulate in the contiguous membranes of the endoplasmic reticulum and the INM (UL34, M50, and BFRF1) unless the nuclear protein UL31 and UL31-related proteins (M53 and BFLF2) arrest the viral membrane protein in the INM. Apparently, complex formation by the two proteins is pivotal for the nuclear egress of herpesvirus nucleocapsids. The nuclear egress complex (NEC) executes this task by interacting with cellular proteins which are already residing in or recruited to the INM and presumably recruits the viral capsids to the egress sites. These interactions lead to the displacement of the rigid nuclear lamina along with nucleocapsid budding (7, 10).In herpes simplex virus type 1 (HSV-1), the interaction between the UL34 and UL31 gene products is required for function. To study the function of the betaherpesvirus NEC in more detail, we started with an analysis of M50/p35 (3). M50/p35, an essential protein, is localized at the nuclear envelope in MCMV-infected cells and recruits its viral interaction partner M53/p38 and cellular protein kinase C to the INM. We identified the M53/p38 binding...

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 ...

The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-α will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-α. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-α induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3.

Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ϳ100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.Our understanding of herpesvirus morphogenesis is mainly derived from studies of Alphaherpesvirinae, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV). A faster replication cycle and a more productive infection in tissue culture aided genetic analysis of alphaherpesvirus morphogenesis. In addition, deletion mutants of key morphogenesis genes in alphaherpesviruses often maintain basic replication capacity, whereas the mutations of their homologues in Betaherpesvirinae or Gammaherpesvirinae mostly result in a lethal phenotype (for the UL31 and the UL34 family, see references 3, 6, 9-11, 16, 20, 21, and 42). These genes became amenable to comprehensive genetic analysis in betaherpesviruses only after their genomes were cloned as infectious bacterial artificial chromosomes (BACs), which obviated the need to generate replication-competent intermediates or complementing cell lines (3, 21, 23). BAC-based mutagenesis allowed viability screens mapping essential genes (8, 43) or even functional sites of essential genes in cytomegaloviruses (3, 21). However, these approaches cannot easily be applied to reveal the null phenotypes in the context of virus replication, as mutant viruses are not easily reconstituted. In addition, deletion of an essential viral gene can reveal the null phenotype of only the first of perhaps several essential functions during virus morphogenesis. This problem can be addressed to some extent by using dominant negative (DN) mutations (36). DN mutants are loss-of-function mutants that induce a null phenotype in the presence of the wild-type (wt) allele (14). Analysis of phenotypes induced by DN mutants proved to be extremely useful in genetics and cell biology, signaling, and biochemistry. Such inhibitory mutants of cellular proteins are often designed based on knowledge on the structural or functional role of a well-characterized protein domain. Unfortunately, we lack the structural information that would allow knowledge-based design of viral DN mutants for the majority of herpesvirus gene products. Thus, we established a random screen consisting of three steps to identify mutants of viral genes with DN potential (36): (i) a library of mutants is generated by random insertion of 5 amino acids (aa) ...