Uncoupling Uncoating of Herpes Simplex Virus Genomes from Their Nuclear Import and Gene Expression

Abstract

Incoming capsids of herpes simplex virus type 1 (HSV-1) enter the cytosol by fusion of the viral envelopes with host cell membranes and use microtubules and microtubule motors for transport to the nucleus. Upon docking to the nuclear pores, capsids release their genomes into the nucleoplasm. Progeny genomes are replicated in the nucleoplasm and subsequently packaged into newly assembled capsids. The minor capsid protein pUL25 of alphaherpesviruses is required for capsid stabilization after genome packaging and for nuclear targeting of incoming genomes. Here, we show that HSV-1 pUL25 bound to mature capsids within the nucleus and remained capsid associated during assembly and nuclear targeting. Furthermore, we tested potential interactions between parental pUL25 bound to incoming HSV-1 capsids and host factors by competing for such interactions with an experimental excess of cytosolic pUL25. Overexpression of pUL25, GFPUL25, or UL25GFP prior to infection reduced gene expression of HSV-1. Electron microscopy and in situ hybridization studies revealed that an excess of GFPUL25 or UL25GFP prevented efficient nuclear import and/or transcription of parental HSV-1 genomes, but not nuclear targeting of capsids or the uncoating of the incoming genomes at the nuclear pore. Thus, the uncoating of HSV-1 genomes could be uncoupled from their nuclear import and gene expression. Most likely, surplus pUL25 competed with important interactions between the parental capsids, and possibly between authentic capsid-associated pUL25, and cytosolic or nuclear host factors required for functional interaction of the incoming genomes with the nuclear machinery.Herpes simplex virus type I (HSV-1) is the most thoroughly studied pathogen among the alphaherpesviruses and among the eight human-pathogenic herpesviruses. Its double-stranded DNA genome of 152 kb is packaged into a capsid that is surrounded by 33 capsid-associated and tegument proteins, which in turn are wrapped by a viral envelope (43,51,68,73,93). HSV-1 assembly commences in the nucleus with the formation of spherical, scaffold-containing procapsids that develop into three types of angularized icosahedral capsids characterized by different sedimentation coefficients, morphologies, protein compositions, and mechanical properties (A, B, and C capsids) (3, 28, 75). The C capsids, which are the heaviest, enclose the viral genome; the B capsids still contain scaffold remnants but no DNA; and the A capsids are empty and lack both scaffold and DNA. After degradation of the scaffold proteins VP21 and VP22a by viral proteases, the procapsids package the viral DNA and mature into C capsids. Predominantly, C capsids leave the nucleus by primary envelopment and de-envelopment at nuclear membranes and acquire their final envelope by secondary envelopment in the cytoplasm (51,52,92,93). B and A capsids most likely result from a failure to initiate or to complete DNA packaging properly and are considered to be dead-end products that are formed during assembly (3, 85, 92).The major capsid prote...

The C Terminus of the Large Tegument Protein pUL36 Contains Multiple Capsid Binding Sites That Function Differently during Assembly and Cell Entry of Herpes Simplex Virus

“…12b) was similar to that after synchronous infection (Fig. 5Dj), demonstrating that cell entry experiments using MOIs of 50, as we had reported previously (26,27,65,100,106), represent cell entry conditions similar to those that occur in a plaque assay.…”

“…5D). After 15 min, the parental capsids were randomly distributed over the entire cytoplasm as described before (26,27,65,100,106), and pUL36 colocalized with capsids (Fig. 5Dc, yellow) but not with gD (Fig.…”

“…The cells were either fixed and permeabilized by incubating them for 4 min in water-free methanol at Ϫ20°C (data not shown) or fixed with 3% (wt/vol) paraformaldehyde in PBS for 20 min and subsequently permeabilized with 0.1% Triton X-100 for 5 min (100, 106). The specimens were labeled with antibodies as described before (27,86,100,106). To block unspecific antibody interactions with the abundantly expressed HSV1 Fc receptor (28) and thus to reduce antigen-independent antibody labeling, we used 10% (vol/vol) human serum of a healthy HSV1-seronegative volunteer and 0.5% (wt/vol) BSA in PBS as blocking reagent (26).…”

“…With its N-terminal binding sites for the tegument proteins VP16 and pUL37 and its C-terminal binding sites for pUL25, it serves as a reversible, multivalent cross-linker between the capsid and the tegument during assembly (16,52,53,74,98,114). pUL25 is a minor structural protein located on the capsid surface that is required for the stable retention of progeny viral genomes within the capsids, nuclear egress, and the release of incoming genomes at the nuclear pores (17,70,92,95,96,100,108,111). In cells infected with HSV1 ⌬UL36 or a mutant encoding only the first 361 aa of pUL36, capsids reach the cytosol, but there is no secondary envelopment, no cell egress, and no plaque formation (25,98).…”

“…The motor protein dynein then propels the cytosolic capsids along microtubules toward the nucleus, where they dock with one capsid corner facing the nuclear pore and release the viral genome into the nucleoplasm for viral transcription and replication (27,40,41,64,65,67,90,100,101,106). At least some HSV1 and PrV pUL36 remains associated with incoming capsids until arrival at the nucleus (4,18,40,62,81,83).…”