Membrane uncoating of intact enveloped viruses

Abstract

Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously "uncoated" with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.

FIG. 1.

Intact (“early-harvest”) and damaged (“late-harvest”) HVJ (Sendai virus). (A) Thin section of late-harvest HVJ (Sendai virus). Arrows indicate the early-harvest viruses present among the viruses in a late-harvest preparation. (B) Glutaraldehyde-fixed and negatively stained early-harvest virus. (C) Thin sections of early-harvest HVJ. Arrow shows virus sectioned at right angles to the long axis. Scale bars, 200 nm. Reproduced from reference with permission of the publisher.

FIG. 2.

Membrane fusion and uncoating of damaged and intact Sendai virus. (A) Damaged Sendai virus that has fused with the membrane of a chorioallantoic cell after 1 h at 4°C and 6 min at 37°C. (B) Damaged Sendai virus that has fused with the membrane of a chorioallantoic cell after 1 h at 4°C and 5 min at 37°C. Much of the viral membrane has merged into the host membrane, and a lot of the viral contents have mixed with the cell cytoplasm. (C) Intact Sendai viruses and mouse strain L cell after 1 h at 4°C and 5 min at 37°C. Note the size and shape of the virus and the organized appearance of the ribonucleoprotein, which is consistent with that of intact viruses. The virus on the right is attached. The virus on the left has fused with the cell and RNP can be seen moving into the cell, but the virus membrane maintains its shape. Scale bars, 100 nm. Reproduced from reference with permission.

FIG. 3.

Model of binding, fusion, and membrane uncoating of intact parainfluenza viruses. (A) Early-harvest parainfluenza virus is attached to a host cell. (B) The viral and host membranes are fused, and a connecting structure is made. Small molecules can diffuse between the cell cytoplasm and virus interior. (C) There is no diffusion of viral membrane glycoproteins into the host membrane. The RNP end traverses the connecting structure to reach the cell. A host structure probably associates with the RNP. (D) The entire RNP reaches the cell interior. As-yet-undefined cytoplasmic structures are thought to be involved in the trafficking of the virus internal contents and participating in viral replication. Viral membrane and glycoproteins are red. Viral RNP is green. Cellular membranes and receptors are blue. Host structures are gray and beige.

FIG. 4.

Membrane fusion and uncoating of an alphavirus (Sindbis virus) at pH 7.2. (A) A Sindbis virus is attached to a BHK cell. The virus is labeled with virus-specific antiserum and gold bead-conjugated goat anti-rabbit antibodies. The viral electron-dense structure, which is presumably the viral core, is intact in the center of the virus. (B) Sindbis virus is connected to host cell by a connecting structure (white arrow). Electron-dense structure is within the virus. (C) Another view of the connecting structure (bottom arrow) between the virus and cell. (D) Part of the electron-dense material moves toward the connecting structure. (E) A fused virus has lost its electron-dense structure, and there is possibly an RNA strand entering the cell (arrow). (F) Immunolabeling provides identification of viral membrane proteins even when standard viral morphology is no longer evident. Empty pieces of viral membrane are released into the surrounding media. Scale bar, 50 nm. Reproduced from reference with permission of the publisher.