Identification of a Role for the trans -Golgi Network in Human Papillomavirus 16 Pseudovirus Infection

We initiated experiments to examine the infection of monocytes postentry. New data show that human cytomegalovirus (HCMV) DNA is detected in the nucleus beginning only at 3 d postinfection in monocytes, compared with 30 min postinfection in fibroblasts and endothelial cells, suggesting that HCMV nuclear translocation in monocytes is distinct from that seen in other cell types. We now show that HCMV is initially retained in early endosomes and then moves sequentially to the trans-Golgi network (TGN) and recycling endosomes before nuclear translocation. HCMV is retained initially as a mature particle before deenvelopment in recycling endosomes. Disruption of the TGN significantly reduced nuclear translocation of viral DNA, and HCMV nuclear translocation in infected monocytes was observed only when correct gH/gL/UL128-131/integrin/c-Src signaling occurred. Taken together, our findings show that viral binding of the gH/gL/UL128-131 complex to integrins and the ensuing c-Src signaling drive a unique nuclear translocation pattern that promotes productive infection and avoids viral degradation, suggesting that it represents an additional viral evasion/survival strategy.monocytes | viral trafficking | gH/gL/UL128-131 complex | integrins | nuclear translocation pathway H uman cytomegalovirus (HCMV) infection usually results in an asymptomatic lifelong persistent infection in healthy individuals after primary infection (1, 2). Although HCMV infection rarely causes disease in immunocompetent individuals, it can cause severe/ fatal disease in immunocompromised patients, such as congenitally infected neonates, patients with AIDS, and transplant recipients (3-6). HCMV infection is also associated with the development of cardiovascular diseases, including atherosclerosis, restenosis, and transplant vascular sclerosis, as well as some cancers (7-10). This wide range of pathological complications results from viral spread to multiple organs and the broad cellular tropism displayed by HCMV following infection (11).Monocytes are a primary site of persistent HCMV infection, and HCMV-infected monocytes are believed to play a key role in the hematogenous dissemination of the virus to target organs following primary infection (12). For HCMV to infect primary monocytes, it must overcome several biological barriers to successfully mediate viral spread and persistence. For example, monocytes do not initially support de novo HCMV gene expression and viral replication (13), and they have a lifespan of only 1-3 d in circulation under normal homeostatic conditions (14, 15). Understanding how HCMV overcomes these biological barriers following infection may provide clues to the underlying causes of HCMV pathogenesis and persistence. Our laboratory has provided molecular evidence for how HCMV evolved to deal with these biological barriers (16)(17)(18)(19)(20)(21)(22).We have shown that the biological changes in HCMV-infected monocytes are triggered by the binding of gB to EGFR (19) and binding of the gH/gL/UL128-131 complex to β1 and β3 integri...

Section: Discussionmentioning

confidence: 50%

Viral binding-induced signaling drives a unique and extended intracellular trafficking pattern during infection of primary monocytes

Kim

Collins-McMillen

Caposio

et al. 2016

“…The tools and approaches used here may reveal that other viruses also use this trafficking pathway. After this work was completed, another laboratory also implicated the TGN in HPV16 entry (38).…”

Section: Discussionmentioning

confidence: 99%

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

Lipovsky

Popa

Pimienta

et al. 2013

173

273

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/ Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

“…Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34,35).…”

mentioning

confidence: 99%

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

DiGiuseppe

Luszczek

Keiffer

et al. 2016

140

During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.HPV entry | vesicular transport | mitosis | digitonin | nuclear vesicle A major hurdle of DNA viruses during a primary infection is successful navigation of the cytoplasm to deliver the viral genome to the nucleus. Foreign DNA that enters the cytoplasm is susceptible to being sensed by innate immune sensors (1). Not surprisingly, viruses have evolved mechanisms to evade detection by these sensors. For example, herpesviruses and adenoviruses both egress into the cytoplasm, yet protect their viral DNA by keeping it encased in viral capsids until directly transferring it into the nucleus through the nuclear pore complex. In contrast, the capsid is unlikely to protect papillomavirus (PV) genomes while traversing the cytoplasm, because the major capsid protein is lost in the endocytic compartment (2).The PV capsid is composed of two viral proteins, the major capsid protein L1 and the minor capsid protein L2, which enclose a chromatinized, circular, double-stranded DNA genome ∼8 kb in size (3-6). Following primary attachment and internalization, acidification of early endosomes triggers capsid disassembly (7-22). Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34, 35).Currently, when and how the human PV (HPV) genome egresses from the membranous compartment is unclear. Here we present evidence indicating that after the onset of mitosis, the viral genome of HPV type 16 (HPV16), an HPV type...