The cytoplasmic events of HCMV virion maturation and trafficking are only beginning to be elucidated. Several viral proteins are known to be required for virion assembly and egress (4
), and cellular proteins of the endoplasmic reticulum (29
), Golgi apparatus (11
), endosomal recycling complex (14
), multivesicular body (10
) and ESCRT (endosomal sorting complex required for transport) (31
) cellular trafficking pathways have also been implicated in virion maturation.
Our results argue that pUL71 is dispensable for events before cytoplasmic envelopment () but required for correct morphogenesis of the viral assembly compartment (vAC) (6
) and its associated vesicular system (; see Fig. S3
in the supplemental material). In the absence of pUL71, the vAC-associated vesicular structures are enlarged (), similar to those previously observed during infections with pUL97-deficient virus or pharmacological inhibitors of pUL97 (32
). These enlarged structures aberrantly contain LAMP1, a cellular marker of late endosomes/lysosomes (). We observed different LAMP1 localizations in side-by-side analysis of mutant and wild-type viruses. This improper vAC constitution generates progeny that are highly cell associated (), and when virus stocks are prepared by sonication of infected cells, mutant virus preparations are less infectious on a per genome basis than wild-type virus preparations (see Fig. S4
in the supplemental material). Despite the effect on egress, the pUL71-deficient mutant remains competent for cell-to-cell spread (), consistent with the earlier conclusion that envelopment is not required for cell-to-cell spread of HCMV (34
), and supporting the view that multiple, distinct virus trafficking pathways exist (26
). A subset of these pathways may promote cell-to-cell spread independently from extracellular virion release.
UL71 mutant virus particles isolated by sonication of infected cells displayed a unique migration through glycerol-tartrate density gradients (). The distribution of mutant viral genomes and infectivity within the gradient followed this novel migration pattern and differed from that observed for wild-type virus preparations (). Analysis of virion proteins revealed that while all assayed species were in UL71 mutant virus particles, their abundance more closely resembled wild-type virus dense bodies than wild-type virions (). Visualization of the mutant particles released from cells by sonication revealed that they were aggregated and associated with sheaths of membrane, as was the case for wild-type virus dense bodies (). These membranes are likely associated with viral proteins, possibly accounting for the similarity in protein abundance for mutant particles and wild-type dense bodies. Although the aggregates of particles with membrane sheaths are produced by mechanical disruption, this observation demonstrates a fundamental difference between wild-type and mutant particles. Wild-type particles were discernible in both cell-free and cell-associated preparations (), as expected (28
), while mutant virus particles were observed only as aggregates in cell-associated preparations ().
Intracellular trafficking of mutant and wild-type virus particles was monitored by TEM (; see Fig. S5
in the supplemental material). In contrast to cells infected with the wild-type virus, almost no virus particles were present in the cytoplasm of cells infected with mutant virus at 96 hpi (). This may indicate that, in addition to cytoplasmic trafficking of virus particles, pUL71 contributes to nuclear egress of nucleocapsids. We did not observe localization of pUL71 to the nucleus or nuclear rim (; see Fig. S3
in the supplemental material), which suggests that if pUL71 acts to promote translocation of nucleocapsids into the cytoplasm, it does so indirectly. By 144 hpi, mutant-virus-infected cells contained many more cytoplasmic virus particles than wild-type-virus-infected cells (), presumably because many wild-type virus particles were released from infected cells before this time (). Conversely, mutant-virus-infected cells developed large, electron-dense ICIs by 96 hpi (). Large ICIs were evident in wild-type-virus-infected cells at 144 hpi ().
Electron-dense ICIs have been observed previously in TEM analyses of HCMV-infected fibroblasts and characterized as lysosomes (35
). In murine cytomegalovirus (CMV)-infected mice, ICI formation in hepatocytes correlated with a reduction in virus titer relative to the titer from salivary gland cells, where the lysosomal structures were not observed (38
). Similar lysosomal structures formed with the same kinetics during HCMV infection of cultured fibroblasts, leading to the proposal that they were a cell type-specific intrinsic defense against infection. These results fit with our observation of large LAMP1-positive vesicles (), as well as LysoTracker green-positive, acidified structures in BADin
UL71STOP-infected cells ().
Our observations, considered in light of the earlier visualization of lysosomal structures (38
), are consistent with a kinetic model of degradation, wherein the formation of large lysosomal structures occurs earlier during mutant virus infection than during wild-type virus infection, resulting in enhanced degradation of mutant virus particles. This model suggests that the virus particles associated with the periphery of ICIs in BADin
UL71STOP-infected cells () subsequently enter the ICI where they are degraded. Trafficking to these lysosomal structures during wild-type virus infection is presumably avoided by rapid, efficient egress before their formation occurs. In the case of mutant virus infection, virion trafficking is inefficient and lysosomal formation occurs earlier, resulting in a larger proportion of virus particles being degraded by lysosomes or sequestered on their periphery. Alternatively, these structures could be directly involved in the normal egress of virus particles, as smaller ICIs are discernible by 96 hpi in wild-type-virus-infected cells (). While direct fusion of multivesicular body-like collections of virions at the plasma membrane has been documented (11
), it is possible that acidified structures are also utilized for egress. The acquisition of proper membranes would likely be important to efficiently utilize acidified structures for egress, as both pH and lipid composition have been shown to be required for formation of acidified multivesicular liposomes (39
). In the case of UL71 mutant virus infection, acquisition of the proper membrane could be impaired by improper vAC morphogenesis (), resulting in inefficient membrane fusion and impaired budding into acidified structures. While we favor the kinetic model of degradation, further experiments are required to rule out the second model.