The Herpes Simplex Virus 2 UL21 Protein Is Essential for Virus Propagation

Abstract

cHerpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ⌬UL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ⌬UL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ⌬UL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ⌬UL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ⌬UL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ⌬UL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.

The Unusual Fold of Herpes Simplex Virus 1 UL21, a Multifunctional Tegument Protein

“…UL21 may also have a role in cytosolic capsid transport through association with microtubules (21). In the absence of UL21, expression of viral genes is delayed, possibly due to lower mRNA levels (8,10). Finally, capsids of UL21-null HSV-2 are unable to undergo nuclear egress (8), suggesting a potential nuclear function for UL21, which localizes not only to the cytoplasm but also to the nucleus (17,21), specifically the nuclear rim (8,9,16).…”

“…UL21 is important for replication in culture because UL21-null mutants of HSV-1 and pseudorabies virus (PRV) show reductions in titer and small plaques (9,10), whereas HSV-2 cannot replicate without UL21 (8). A lack of UL21 also leads to defects in pathogenesis of PRV in mice and pigs (11)(12)(13)(14).…”

“…Herpes simplex virus 1 (HSV-1) UL21 is a 535-amino-acid tegument protein that is conserved within the Alphaherpesvirus subfamily (8) and may have analogs among other herpesviruses. UL21 is important for replication in culture because UL21-null mutants of HSV-1 and pseudorabies virus (PRV) show reductions in titer and small plaques (9,10), whereas HSV-2 cannot replicate without UL21 (8).…”

Herpes Simplex Virus 2 Virion Host Shutoff Endoribonuclease Activity Is Required To Disrupt Stress Granule Formation

“…Additionally, PCR fragments amplified from each recombinant BAC clone and from DNA isolated from the reconstituted D215N virus were sequenced to verify that recombination had occurred at the correct location within the viral genome, to verify that the D215N mutation was present, and to verify that no additional mutations were present within UL41. Virus was reconstituted from BAC DNA by transfection of Vero cells as described previously (52). A similar strategy was used to repair the D215N mutation (D215NRep) using a product amplified from pRF87 (27) for the initial recombination step.…”

“…pYEbac373, the fulllength infectious HSV-2 186 bacterial artificial chromosome (BAC), was constructed as described previously (52). A BAC carrying full-length vhs with the point mutation D215N was constructed by the two-step Redmediated recombination procedure, using pYEbac373 in Escherichia coli GS1783 (53).…”