Human cytomegalovirus pUS24 is a virion protein that functions very early in the replication cycle

Abstract

We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

FIG. 1.

pUS24-deficient mutants produce reduced yields compared to the wild-type virus. (A) Location of mutations. BADinUS24 contains a transposon insertion after base pair 1164 of the US24 ORF, and BADsubUS24 contains a transposon cassette with 79 base pairs (363 to 441) of the US24 ORF substituted. (B) Western blot analysis of pUS24. Fibroblasts were infected with wild-type (BADwt) and mutant viruses at a multiplicity of infection (MOI) of 1 PFU/cell and analyzed using a pUS24-specific monoclonal antibody at the indicated times. β-Actin was monitored as a loading control. hpi, hours postinfection. (C) pUS24-deficient viruses exhibit a 20- to 30-fold growth defect compared to BADwt after infection of fibroblasts at an MOI that was relatively high (MOI = 1) or low (MOI = 0.01). At the indicated times, medium was collected from infected cultures and virus titers were determined by plaque assay. (D) Mutant viruses produce less cell-free and cell-associated virus. At the indicated times, cell-free and cell-associated virus was collected from cultures of infected fibroblasts, and the virus titer was determined by plaque assay. (E) BADrevUS24, a revertant derived from BADinUS24, exhibits wild-type growth. Infectivity in the medium was assayed by plaque assay at the indicated times. (F) US23 RNA accumulation is not disrupted in the two US24 mutants. US23-specific RNA was assayed by Northern blotting using a strand-specific riboprobe to the US23 ORF (nucleotides 1 to 215). 18S rRNA was monitored as a loading control.

FIG. 2.

pUS24-deficient virus appears normal when compared to wild-type virus after infection with equal numbers of infectious particles. (A) IE1, pUL44, and pp28 are expressed normally after infection with US24-deficient virus. Fibroblasts were mock infected or infected with BADwt or BADinUS24 at a multiplicity of infection of 1 PFU/cell, and the accumulation of viral proteins was assayed by Western blotting after various time intervals. (B) The cellular localizations of IE1, pUL44, and pp28 are normal after infection with US24-deficient virus. Fibroblasts were infected with BADwt or BADinUS24 at a multiplicity of infection of 0.3 PFU/cell, and viral proteins were assayed by immunofluorescence 72 h later (green, GFP; red, IE1, pUL44, or pp28). (C) Viral DNA accumulation is normal in the absence of pUS24. Fibroblasts were mock infected or infected at a multiplicity of infection of 1 PFU/cell, and virus DNA was assayed by slot blot at the indicated times.

FIG. 3.

Electron microscopy shows that virus particles accumulate normally after infection with a pUS24-deficient virus. Fibroblasts were fixed and processed for imaging at 96 h after infection with BADwt or BADinUS24. Arrows identify C capsids in the nucleus and virions in the cytoplasm.

FIG. 4.

Characterization of pUS24-deficient virions. (A, left) A pUS24-deficient mutant has a 10-fold-higher genome/PFU ratio than wild-type virus. The amounts of virus DNA present in 4 × 10⁴ PFU of BADwt and BADinUS24 were determined by real-time PCR. Results are shown for undiluted and diluted (1:10) samples (in duplicate), and a sample with no added virus DNA was analyzed as a control. (A, right) Statistical summary of three independent experimental results. (B) pUS24 is a tegument protein. BADwt and BADinUS24 particles were purified, and equivalent amounts of protein from virions, NIEPs, and DBs were analyzed by Western blotting after no treatment (−), trysin digestion (T), or trypsin digestion after disruption of particles with Triton X-100 (TT). pUS24, pp28, and gB were assayed by Western blotting. (C) Colocalization of pUS24 and pp65 in the cytoplasm of infected fibroblasts. At 72 h after infection with BADwt, the localizations of virus proteins were assayed by immunofluorescence. pUS24 is represented by red, pp65 by green, and DAPI by blue; yellow marks colocalized pUS24 and pp65. (D) The levels of nine virion proteins are the same in BADinUS24 and BADwt virions. Equivalent amounts (normalized for the number of viral genomes) of gradient-purified virions were assayed by Western blotting using antibodies specific for the indicated virus-encoded proteins.

FIG. 5.

The expression of immediate-early protein and RNA is delayed in cells infected with pUS24-deficient virus when cells receive equal numbers of particles. Viral protein (A) and mRNA (B) expression was delayed after infection with BADinUS24 compared to BADwt. Cells received particles containing the same number of mutant or wild-type genomes. At the indicated times IE1, pUL44, and pp28 were assayed by Western blotting and IE1 mRNA was assayed by Northern blotting. β-Actin protein and 18S rRNA were monitored as loading controls. hpi, hours postinfection.

FIG. 6.

The virion proteins pp65 and pp71 move into the nucleus, but IE1 expression is reduced in BADinUS24-infected compared to BADwt-infected cells when cells receive equal numbers of particles. Fibroblasts were infected with aliquots of virus containing equal numbers of PFU or genomes. Cells were fixed and imaged by immunofluorescence at 2 h (pp65 and pp71) or 12 h postinfection (IE1).

FIG. 7.

Viral genomes move to the nucleus in BADinUS24-infected cells as efficiently as in BADwt-infected cells. (A) Separation of nuclear and cytoplasmic fractions. At 2 h postinfection, infected fibroblasts were collected and fractionated. Equal amounts of protein from the total cell lysate, the nuclear fraction, and cytoplasmic fraction were assayed for pp65 and pp28 by Western blotting. (B) Similar amounts of viral DNA accumulate in the nucleus after infection with equal numbers of mutant and wild-type particles. Viral DNA was quantified by real-time PCR analysis using a primer pair specific to the UL123 ORF.