Herpes simplex virus 1 Us3 deletion mutant is infective despite impaired capsid translocation to the cytoplasm

Abstract

Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i) The number of R7041(∆US3) capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii) The mean number of R7041(∆US3) virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii) 98% of R7041(∆US3) virions were in the perinuclear space; (iv) The number of R7041(∆US3) capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3) yields were 2.37×10(8) and HSV-1 yields 1.57×10(8) PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3) virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Figure 1

TEM of a Vero cell 12 hpi with R7041(∆Us3). Many capsids are at the nuclear periphery, some of them bud (b) at the inner nuclear membrane (INM) (i). The INM formed folds that penetrate into the nucleus clearly seen in the inset. Fully enveloped virions are located adjacent to folds (f), and one in the rough endoplasmic reticulum (RER) (v). The outer nuclear membrane (ONM) (o) is only clearly visible in the inset. Bar = 500 nm.

Figure 2

TEM of a Vero cell 24 hpi with R7041(∆Us3). Large number of virions are located in the PNS and in invaginations (f) formed by folding of the INM into the nucleus, and in “vacuoles” often referred to as herniations. They are considered likely to represent invaginations of the INM as easily apparent at the right bottom. A few virions are in the RER (rer) adjacent to the perinuclear space (PNS). Numerous capsids (c) are distributed in the nucleus and some in the cytoplasm. A few capsids bud (b) at the INM. Bar = 500 nm.

Figure 3

Cryo-FESEM of a R7041(∆Us3) infected Vero cell (12 hpi) showing a fracture plane through the nucleus (n) and the cytoplasm (cy). The INM (i) formed invaginations from which folds (f) arise. Membranes of herniations (h) continue into the INM. Capsids (c) bud (b) at the INM. Virions (arrows) are in invaginations and in the PNS. Bar = 500 nm.

Figure 4

Mean and standard deviation of R7041(∆U_S3) and wt HSV-1 capsids budding at the INM, and of virions within the PNS calculated per mean nuclear volume. Budding of R7041(∆U_S3) capsids peaked at 12 hpi whereas the number of R7041(∆U_S3) and wt HSV-1 virions potentially increased (potential trend lines) though with a difference of more than 2000 virions by 24 hpi. p < 0.0001, n = 5.

Figure 5

TEM images of budding capsids (A) at the INM (i) and (B) at the ONM (o). Note the identity of the two phenotypes, the ridge (arrows) formed on one side at each particle, and the eccentricity of the process. Ridges and eccentricity are not consistent with fusion. Bars 200 nm.

Figure 6

(A) Scheme of the envelopment pathways of HSV-1. Capsids bud at the INM (B-INM) and are postulated to fuse with the ONM. However, the process has all characteristic of budding (B-ONM). Capsids in the cytoplasm are transported to the Golgi complex where they bud into cisternae (B-Golgi) or they bud at Golgi membranes in such a manner that concomitantly transport vacuoles are formed (Wrapping). In both cases, the vacuolar membrane is dispatched from the donor membrane by fission. Alternatively, virions are transported from the PNS into RER cisternae. We postulate that they can be further transported into Golgi cisternae via the endoplasmic reticulum Golgi intermediate compartment (ERGIC) that connects the RER with the Golgi complex in herpes virus infected cells. Virions in transport vacuoles are released into the extracellular space by exocytosis. (B) Phenotype distribution of R7041(∆Us3) and HSV-1 envelopment. Mean total number and standard deviation of virus particles expressed per mean nuclear volume of budding capsids at the INM (B-INM) and of virions in the PNS (shown in more detail in Figure 4), and of capsids budding at the ONM (B-ONM). Virions in the RER, capsids in the cytoplasm (Capsids), capsids budding at Golgi membranes (B-Golgi), virions derived by wrapping (Wrapping), and virions in Golgi cisternae or vacuoles (Golgi) were expressed per mean cell volume. Data from R7041(∆Us3) infected cells are significantly different compared to wt HSV-1 infected cells (* p < 0.001, n = 5).

Figure 7

TEM images of Golgi fields in Vero cells at 12 hpi with wt HSV-1(A) and at 20 hpi (B) with the repair mutant R2641. (A) Close to a large Golgi stack is a virion in a concentric vacuole (Vw) derived by wrapping. Typically, the space between viral envelope and vacuolar membrane is similar in electron density as the tegument. In contrast, the space between viral envelope and membrane of vacuoles or Golgi cisternae containing one or more virions (arrow) derived either by budding into it or by intraluminal transportation from the RER. The space between virions and vacuolar membrane is electron lucent. (c) capsids, (b) budding capsid. Note that the outermost membrane of the Golgi stack shows the characteristic of RER membranes (rer) indicating connectivity between Golgi complex and RER. (B) Three vacuoles or Golgi cisternae (arrows) close to a small Golgi stack contain virions derived by budding or by intraluminal transportation. One virion is in the RER (Ve), and one capsid in the cytoplasm. Bar = 500 nm.

Figure 8

TEM images of R7041(∆U_S3) infected Vero cells at 12 hpi showing three Golgi fields (go) with thick electron dense membranes, and a concentric vacuole enclosing a virion that resulted from wrapping (Vw). There are virions (Vp) in the PNS, and one capsid (c) in the cytoplasm. Bar = 500 nm.

Figure 9

TEM images at 24 hpi with R7041(∆U_S3). Multiple Golgi stacks (go) containing thick electron dense membranes, a single capsid (c) in the cytoplasm and one in the process of wrapping (w), virions in the PNS (Vp) and capsids budding (b) at the INM. Bar = 500 nm.

Figure 10

TEM images at 20 hpi with R7041(∆U_S3) showing an area with at least 13 capsids (c), multiple Golgi stacks (go) containing thick electron dense membranes, and a capsid in the process of wrapping (w) that results in a malformed virion. One capsids bud (b) at a membrane that cannot be identified. Bar = 500 nm.

Figure 11

TEM images of three Golgi fields (go) containing thick electron dense membranes at 24 hpi with R7041(∆U_S3). One virion has probably entered the cisterna by budding or via intraluminal transportation (Vg), the other virions has been probably derived by wrapping (Vw). Note the difference in electron density of the space between viral envelope and vacuolar membrane. Bar = 500 nm.

Figure 12

One-Step growth curves of R7041(∆Us3) and wt HSV-1. Cells were harvested at times post inoculation as indicated. Infectious virus yields in pellets and supernatants were determined by plaque titration. Mean and standard deviation were calculated from 4 experiments.

Figure 13

Mean total number and standard deviation of R7041(∆Us3) and wt HSV-1 virus particles (capsids in the cytoplasmic matrix, budding capsids at any membrane as well as virions), and of virions including those in the PNS, RER, Golgi cisternae and/or vacuoles. (* p < 0.001, n = 5).