Tetrahalogenated benzimidazole D-ribonucleosides are active against rat cytomegalovirus.

BACKGROUND: Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the viral terminase, the enzyme complex responsible for viral DNA cleavage into single unit-length genomes and subsequent DNA packaging into procapsids. Here, we evaluated the viral inhibition by benzimidazole D-ribonucleosides against rat cytomegalovirus (RCMV). METHODS: Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by measurement of plaque formation. Yield assays and electron microscopy of thin sections was performed using RCMV-infected cells in the presence or absence of the compounds. The effects of Cl4RB and BTCRB on cleavage of concatemers was analyzed by pulsed-field gel electrophoresis. To characterize the behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model was employed where cells are embedded in an extracellular matrix using rat-tail collagen I. RESULTS: Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed that only few virions were formed in RCMV-E infected cells in the presence of the compounds. Pulsed-field gel electrophoresis showed that DNA concatemers failed to be processed in the presence of the compounds. Yield Assays showed a comparable viral growth in the 3D vs. 2D cell culture as well as inhibition in the presence of Cl4RB or BTCRB for RCMV-E/GFP. CONCLUSIONS: These results demonstrate that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV-E by preventing cleavage of concatemeric DNA and nuclear egress of mature capsids.

Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM.

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 Å resolution and describe the molecular interactions among the ∼3000 protein molecules in the MCMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and ß-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a "saddle loop" region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.

Quantitative investigation of murine cytomegalovirus nucleocapsid interaction.

In this study, we quantitatively investigate the role of the M97 protein for viral morphogenesis in murine cytomegalovirus (MCMV)-infected fibroblast cells. For this purpose, a statistical analysis is performed for the spatial distribution of nuclear B-capsids (devoid of DNA, containing the scaffold) and C-capsids (filled with DNA). Cell nuclei infected with either wild-type or an M97 deletion mutant were compared. Univariate and multivariate point process characteristics (like Ripley's K-function, the L-function and the nearest neighbour distance distribution function) are investigated in order to describe and quantify the effects that the deletion of M97 causes to the process of DNA packaging into nucleocapsids. The estimation of the function L(r) -r reveals that with respect to the wild type there is an increased frequency of point pairs at a very short distance (less than approximately 100 nm) for both the B-capsids as well as for the C-capsids. For the M97 deletion mutant type this is no longer true. Here only the C-capsids show such a clustering behaviour, whereas for B-capsids it is almost nonexistant. Estimations of functionals such as the nearest neighbour distance distribution function confirmed these results. Thereby, a quantification is provided for the effect that the deletion of M97 leads to a loss of typical nucleocapsid clustering in MCMV-infected nuclei.

Cytomegalovirus primary envelopment occurs at large infoldings of the inner nuclear membrane.

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.

Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection.

A recombinant murine cytomegalovirus (mCMV) that expresses enhanced green fluorescent protein (EGFP) under control of the native immediate-early 1/3 promoter was constructed to detect directly sites of viral activity in latent and reactivated infections. The recombinant virus had acute and latent infection characteristics similar to those of wild-type mCMV. Rare green-fluorescing foci were observed in paraffin sections from lungs and spleens infected latently. Positive immunoperoxidase staining for EGFP in sections of the same lung tissues suggests that these cells may be sites of restricted viral gene expression. EGFP was detected easily in tissue explants reactivating from latent infection in vitro. Morphology and adhesion characteristics of fluorescing cells suggest that viral reactivation occurs in tissue macrophages in explant cultures. The observations presented in this study demonstrate the usefulness of EGFP-expressing recombinants as tools for direct tracking of mCMV activity in vivo and in vitro.

The murine cytomegalovirus M25 open reading frame encodes a component of the tegument.

The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse-chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a lambdagt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643-650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.