Jens von Einem

The Tegument Protein UL71 of Human Cytomegalovirus Is Involved in Late Envelopment and Affects Multivesicular Bodies

ABSTRACT Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.

A new tool linking human cytomegalovirus drug resistance mutations to resistance phenotypes

Drug resistant strains of human cytomegalovirus (HCMV) in patients at risk may increasingly develop into a problem in the clinical setting. Genotypic resistance testing is becoming the method of choice, but requires previous phenotypic characterisation of each newly found mutation. In order to facilitate the interpretation of the patients CMV sequence data, a web-based search tool was generated that links the sequence to a database containing all published UL97 (protein kinase) and UL54 (DNA polymerase) mutations and corresponding antiviral drug susceptibility phenotypes. It is reasonable to assume that HCMV drug resistance testing will provide relevant data for an adjustment of therapy and on prognosis of clinical outcome. HCMV drug susceptibility testing will become even more important once new drugs will be available for therapy allowing a wider choice of antiviral agents to treat HCMV disease. These topics will also play a pivotal role for optimising antiviral therapy of HCMV- and other viral diseases.

A molecular tweezer antagonizes seminal amyloids and HIV infection

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a ‘molecular tweezer’ specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion–amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses. DOI: http://dx.doi.org/10.7554/eLife.05397.001

Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy

Electron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. High‐pressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three‐dimensional visualization after serial sectioning. For elucidation of dynamic processes three‐dimensional tomography is extremely helpful. We analysed interaction of HCMV particles with host cell membranes during final envelopment. Both wild‐type virus and a viral mutant with impaired envelopment were analysed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) – a virus‐induced cytosolic structure – by using three‐dimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.

M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

ABSTRACT The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness

ABSTRACT One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator gene, enhanced green fluorescence protein was C-terminally fused to the HCMV early-late protein UL83 (pp65) or the late proteins UL32 (pp150) and UL99 (pp28), resulting in reporter viruses vTB65g, vTB150g, and vTB28g. vTB65g proved to be superior to the other constructs due to its favorable signal-to-noise ratio and was therefore used to establish the optimum conditions for our assay. The UL54 E756K and D413E mutations were introduced into vTB65g by markerless bacterial artificial chromosome mutagenesis, resulting in virus strains vE756Kg and vD413Eg. The drug susceptibility phenotypes of vE756Kg and vD413Eg were comparable to those previously reported. Furthermore, we found a reduced replicative fitness of vE756Kg by measuring fluorescence intensity as well as by conventional virus growth kinetics. Decreased fluorescence signals of vE756Kg- and vD413Eg-infected cells at late times of infection suggested a reduced polymerase activity, which was confirmed by real-time PCR quantification of the newly synthesized viral DNAs. This new fluorescence-based assay is a highly reproducible method for the phenotypic characterization of mutations potentially influencing drug susceptibility, viral replicative fitness, and polymerase activity of HCMV after marker transfer.

A Beta-Herpesvirus with Fluorescent Capsids to Study Transport in Living Cells

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion.

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins

Streptolysin O (SLO) is a cholesterol‐dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca2+ signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO‐induced Ca2+ signalling remain poorly understood. Here, we show that in NCI‐H441 cells, wild‐type SLO as well as non‐pore‐forming mutant induces long‐lasting intracellular Ca2+ oscillations via IP3‐mediated depletion of intracellular stores and activation of store‐operated Ca2+ (SOC) entry. SLO‐induced activation of SOC entry was confirmed by Ca2+ add‐back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca2+ oscillations were comparatively short‐lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short‐lived oscillatory response into a long‐lived one. Thus, we conclude that SLO‐mediated Ca2+ signalling involves Ca2+ release from intracellular stores and STIM1/Orai1‐dependent SOC entry. The phenotype of Ca2+ signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry‐associated proteins in S. pyogenes‐induced lung infection and pneumonia.

The molecular tweezer CLR01 inhibits Ebola and Zika virus infection

ABSTRACT Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re‐emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV‐1 and herpes viruses through a selective interaction with lipid‐raft‐rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose‐dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad‐spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent. HighlightsThe molecular tweezer CLR01 inhibits EBOV and ZIKV infection.ZIKV infection of cells derived from the anogenital tract and the central nervous system is blocked.Anti‐ZIKV activity is retained in semen, urine, saliva, and CSF.CLR01 represents a broad‐spectrum inhibitor of enveloped viruses with prospects as a microbicide or antiviral agent.

Semen inhibits Zika virus infection of cells and tissues from the anogenital region

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.Semen from Zika virus infected individuals can contain high viral loads and can result in sexual transmission. Here, Müller et al. show that semen, and particularly seminal preparations containing extracellular vesicles, inhibit infection of Zika and other flaviviruses.