The ESCRT machinery is not required for human cytomegalovirus envelopment.
The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.
Fraile-Ramos
Alberto
A
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autonoma, Madrid 28049, Spain. alberto.fraile@cnb.uam.es
Pelchen-Matthews
Annegret
A
Risco
Cristina
C
Rejas
María T
MT
Emery
Vincent C
VC
Hassan-Walker
Aycan F
AF
Esteban
Mariano
M
Marsh
Mark
M
D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics; D015703:Antigens, CD / Q000032:analysis; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D003587:Cytomegalovirus / Q000254:growth & development / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000235:genetics / Q000502:physiology* / Q000821:virology*; D020868:Gene Silencing; D051528:Guanylate Kinases; D006801:Humans; D016253:Microscopy, Immunoelectron; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D060149:Tetraspanin 30; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D014771:Virion / Q000737:chemistry; D019065:Virus Assembly
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; C554586:CD63 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D060149:Tetraspanin 30; D033921:Vesicular Transport Proteins; D051528:Guanylate Kinases; C485777:MAGI2 protein, human; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human; C490304:VPS4B protein, human