Sonja Welsch

Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites

Summary Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.

HIV-1 Antagonism of CD317 Is Species Specific and Involves Vpu-Mediated Proteasomal Degradation of the Restriction Factor

Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.

Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

New methodology improves the spatial resolution and sensitivity of correlative light and EM tomography, revealing new insights into dynamic cellular processes.

HIV-1 Buds Predominantly at the Plasma Membrane of Primary Human Macrophages

HIV-1 assembly and release are believed to occur at the plasma membrane in most host cells with the exception of primary macrophages, for which exclusive budding at late endosomes has been reported. Here, we applied a novel ultrastructural approach to assess HIV-1 budding in primary macrophages in an immunomarker-independent manner. Infected macrophages were fed with BSA-gold and stained with the membrane-impermeant dye ruthenium red to identify endosomes and the plasma membrane, respectively. Virus-filled vacuolar structures with a seemingly intracellular localization displayed intense staining with ruthenium red, but lacked endocytosed BSA-gold, defining them as plasma membrane. Moreover, HIV budding profiles were virtually excluded from gold-filled endosomes while frequently being detected on ruthenium red–positive membranes. The composition of cellular marker proteins incorporated into HIV-1 supported a plasma membrane–derived origin of the viral envelope. Thus, contrary to current opinion, the plasma membrane is the primary site of HIV-1 budding also in infected macrophages.

Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses

Macrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4(+) T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4(+) T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1-infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4+ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.

More than one door – Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.

Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

Ultrastructural analysis of a filovirus assembling within infected eukaryotic cells reveals differences in structure and assembly mechanisms between related RNA viruses.

HIV-1 Gag Processing Intermediates Trans-dominantly Interfere with HIV-1 Infectivity

Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations. To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

Ultrastructural Analysis of ESCRT Proteins Suggests a Role for Endosome‐Associated Tubular–Vesicular Membranes in ESCRT Function

The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV‐budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular–vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV‐infected cells.