The envelope glycoproteins of primate lentiviruses, including human and simian immunodeficiency viruses (HIV and SIV), are heterodimers of a transmembrane glycoprotein (usually gp41), and a surface glycoprotein (gp120), which binds CD4 on target cells to initiate viral entry. We have used electron tomography to determine the three-dimensional architectures of purified SIV virions in isolation and in contact with CD4+ target cells. The trimeric viral envelope glycoprotein surface spikes are heterogeneous in appearance and typically ∼120 Å long and ∼120 Å wide at the distal end. Docking of SIV or HIV-1 on the T cell surface occurs via a neck-shaped contact region that is ∼400 Å wide and consistently consists of a closely spaced cluster of five to seven rod-shaped features, each ∼100 Å long and ∼100 Å wide. This distinctive structure is not observed when viruses are incubated with T lymphocytes in the presence of anti-CD4 antibodies, the CCR5 antagonist TAK779, or the peptide entry inhibitor SIVmac251 C34. For virions bound to cells, few trimers were observed away from this cluster at the virion–cell interface, even in cases where virus preparations showing as many as 70 envelope glycoprotein trimers per virus particle were used. This contact zone, which we term the “entry claw”, provides a spatial context to understand the molecular mechanisms of viral entry. Determination of the molecular composition and structure of the entry claw may facilitate the identification of improved drugs for the inhibition of HIV-1 entry.

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.

Understanding the molecular architectures of enveloped and complex viruses is a challenging frontier in structural biology because in most cases, the structural and compositional variation from one viral particle to another precludes the use of either crystallization or image averaging procedures that have been successfully implemented in the past for highly symmetric viruses. While advances in cryo electron tomography of unstained specimens provide new opportunities for identification and molecular averaging of individual subcomponents such as the surface glycoprotein spikes on these viruses, electron tomography of stained and plunge-frozen cells is being used to visualize the cellular context of viral entry and replication. Here, we review recent developments in both areas as they relate to our understanding of the biology of heterogeneous and pleiomorphic viruses.

Background: Infant death in KID syndrome is recognized; its association with specific genotypes and pathophysiology is inadequately understood. Objective: To discover characteristics that account for poor outcomes in lethal KID syndrome. Methods: We collected four new cases and nine previously reported, genotyped cases of lethal KID syndrome. We performed new molecular modeling of the lethal mutants GJB2 p.A88V and GJB2 p.G45E. Results: Infant death occurred in all patients with GJB2 p.G45E and p.A88V; it is unusual with other GJB2 mutations. Early death with those two "lethal" mutations is likely multifactorial: during life all had at least one serious infection; most had poor weight gain and severe respiratory difficulties; many had additional anatomic abnormalities. Structural modeling of GJB2 p.G45E identified no impact on the salt bridge previously predicted to account for abnormal central CO 2 sensing of GJB2 p.A88V. Limitations: Clinical review was retrospective. Conclusion: GJB2 p.G45E and p.A88V are the only KID syndrome mutations associated with uniform early lethality. Those electro-physiologically severe mutations in GJB2 reveal abnormalities in many organs in lethal KID syndrome. All KID syndrome patients may have subtle abnormalities beyond eyes, ears and skin. Early genotyping of KID syndrome births will inform prognostic discussion.