The pp24 phosphoprotein of Mason-Pfizer monkey virus contributes to viral genome packaging

BACKGROUND: The Gag protein of Mason-Pfizer monkey virus, a betaretrovirus, contains a phosphoprotein that is cleaved into the Np24 protein and the phosphoprotein pp16/18 during virus maturation. Previous studies by Yasuda and Hunter (J. Virology. 1998. 72:4095–4103) have demonstrated that pp16/18 contains a viral late domain required for budding and that the Np24 protein plays a role during the virus life cycle since deletion of this N-terminal domain blocked virus replication. The function of the Np24 domain, however, is not known. RESULTS: Here we identify a region of basic residues (KKPKR) within the Np24 domain that is highly conserved among the phosphoproteins of various betaretroviruses. We show that this KKPKR motif is required for virus replication yet dispensable for procapsid assembly, membrane targeting, budding and release, particle maturation, or viral glycoprotein packaging. Additional experiments indicated that deletion of this motif reduced viral RNA packaging 6–8 fold and affected the transient association of Gag with nuclear pores. CONCLUSION: These results demonstrate that the Np24 domain plays an important role in RNA packaging and is in agreement with evidence that suggests that correct intracellular targeting of Gag to the nuclear compartment is an fundamental step in the retroviral life cycle

Human Ubc9 Contributes to Production of Fully Infectious Human Immunodeficiency Virus Type 1 Virions

Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, trafficking, and Env incorporation. The Gag-mediated assembly of virus particles and protease-mediated processing of Gag and Gag-Pol were unchanged in the absence of Ubc9. However, the stability of the cell-associated Env glycoprotein was decreased and Env incorporation into released virions was altered. Interestingly, overexpression of the Ubc9 trans-dominant-negative mutant C93A, which is a defective E2-SUMO-1 conjugase, suggests that this activity may not be required for interaction with Gag, virion assembly, or infectivity. This finding demonstrates that Ubc9 plays an important role in the production of infectious HIV-1 virions

Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy

Positive correlation between Merkel cell polyomavirus viral load and capsid-specific antibody titer

Merkel cell polyomavirus (MCPyV or MCV) is the first polyomavirus to be clearly implicated as a causal agent underlying a human cancer, Merkel cell carcinoma (MCC). Infection with MCPyV is common in the general population, and a majority of adults shed MCPyV from the surface of their skin. In this study, we quantitated MCPyV DNA in skin swab specimens from healthy volunteers sampled at different anatomical sites over time periods ranging from 3 months to 4 years. The volunteers were also tested using a serological assay that detects antibodies specific for the MCPyV virion. There was a positive correlation between MCPyV virion-specific antibody titers and viral load at all anatomical sites tested (dorsal portion of the hands, forehead, and buttocks) (Spearman’s r 0.644, P < 0.0001). The study results are consistent with previous findings suggesting that the skin is primary site of chronic MCPyV infection in healthy adults and suggest that the magnitude of an individual’s seroresponsiveness against the MCPyV virion generally reflects the overall MCPyV DNA load across wide areas of the skin. In light of previous reports indicating a correlation between MCC and strong MCPyV-specific seroresponsiveness, this model suggests that poorly controlled chronic MCPyV infection might be a risk factor in the development of MCC

The Role of Human Ubc9 During the Human Immunodeficiency Virus Replication Cycle

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus and the causative agent of the acquired immune deficiency syndrome (AIDS) pandemic. The retrovirus replication cycle is divided into early infectious events, which involve the infection and integration of the viral DNA into target cell chromosomes; and late events, which involve the expression of viral genes and assembly of infectious virions. To complete the replication cycle, HIV-1 utilizes various cellular pathways. We identified the Ubc9 E2 SUMO conjugating enzyme as a HIV-1 Gag interaction partner. When this interaction was disrupted in HIV-1 producer cells by Ubc9 siRNA, the virus that was released from the cells was approximately 10-fold less infectious than virus released from control cells. The decreased virion infectivity was found to be due to decreased levels of intracellular mature Env, which in turn decreased the amount of Env packaged into assembling virions. Surprisingly, the defect in mature Env levels in Ubc9 knockdown cell was dependent upon Gag expression. The mechanism of decreased mature Env in Ubc9 knockdown cells was examined and we found that gp120/gp41 appeared to be preferentially degraded at a post gp160 cleavage step, but before transport/association with the plasma membrane and lipid rafts. The intracellular gp120 levels were restored when cells were treated with lysosome inhibitors, which suggested that the decreased intracellular Env stability is due to increased lysosomal degradation; however, inhibiting Env degradation did not restore Env packaging into assembled virions. Since lysosomal degradation is linked with autophagy, we examined autophagy in Ubc9 knockdown cells and found that autophagy was increased approximately 3-fold compared to control cells. Autophagy inhibitors were unable to block autophagy, and we were unable to determine if increased lysosomal degradation of gp120 was occurring through an autophagy dependent or independent mechanism. We have also examined the potential role of Ubc9 during the HIV-1 early infectious events. Decreased LTR driven gene expression was detected following the infection of Ubc9 knockdown cells with VSV-G pseudotyped HIV-1 virions. These results suggested that Ubc9 plays a functional role during the early events of the HIV-1 replication cycle at a post-entry step. Adviser: Charles Woo

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions

HIV-1 Gag and Env exhibit altered association with lipid rafts in Ubc9 knockdown cells.

<p>(a) Lipid raft isolation. Representative immunoblot of sucrose fractions for lipid raft markers Flotillin-1 and non-raft marker transferrin receptor 1 (TfR). Fractions containing lipid raft markers (LR), detergent soluble membranes (DSM; raft excluded), and detergent resistant membranes (DRM; non DSM). (b) Average density of all sucrose fractions prior to being adjusted to 1X lysis buffer. 293T cells were transfected with pNL4-3 alone (c), or in combination with either Ctr. siRNA (d) or Ubc9 siRNA (e). Cells were labeled with [³⁵S] methionine/cysteine for 4 hours. Cells were placed on ice and transferred to a 4°C cold room where they were lysed with ice cold TNE buffer for 30 minutes on ice followed by homogenization with a 25G needle. Lysates were clarified in a cooled micro centrifuge and then adjusted to 60% sucrose and overlaid with a discontinuous sucrose gradient and centrifuged at 100,000 X g at 4C for at least 18 hrs. Gradients were fractioned into 11 equal samples using a Biocomp piston fractionator and adjusted to 1x lysis buffer. Viral proteins were immunoprecipitated with patient serum, separated by SDS PAGE, and visualized by phosphorimaging using The Discovery Series Quantity One software. Lipid raft floatation experiments were carried out in triplicate. Representative over-exposed gels are shown so that viral proteins associated with lipids rafts can be more easily visualized. (f) Immunoblot of transfected cell lysates; untransfected (lane 1), NL4-3 + Ctr. siRNA (lane 2), NL4-3 + Ubc9 siRNA (lane 3). (g) Ratio of gp120/Pr55 proteins associated with lipid rafts. (h) Percent of total cellular Gag associated with lipid rafts.</p

Stability of gp160 is unchanged in Ubc9 knockdown cells.

<p>(a) 293T cells were transfected with pNL4-3 MUT 511 alone, or in combination with Ctr. siRNA or Ubc9 siRNA, or left untransfected. Cells were pulse (P) labeled with [³⁵S] methionine/cysteine for 1 hour, then chased for 2 and 4 hours. Samples were processed as described in previous Endo H _f experiment. A representative over-exposed gel is shown so that partially Endo H_f resistant Env can be more easily visualized. The identity of Endo H_f, untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo H_f sensitive forms of gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo H_f resistant forms of gp160 that have undergone glycan modification in the TGN are labeled as gp160r. These forms of gp160 denoted as gp160* in Endo H_f untreated samples. Lower panel: Immunoblot of Ubc9 expression in whole cell lysates; untransfected (group 1), pNL4-3 (group 2), Ctr. siRNA (group 3), and Ubc9 siRNA (group 4). (b) Furin activity is not dependent upon Ubc9 expression. 293T cells were lysed 24 hrs post transfection and cell lysates were immunoblotted with antibodies against Ubc9, Actin, and E-Cadherin. Immature, uncleaved E-Cadherin is designated as pro E-Cadherin.</p

Working model of Env trafficking through the cell in Ubc9 knockdown cells.

<p>HIV-1 Env trafficking from the ER to the TGN occurs normally. After cleavage into gp120/gp41 and transport out of the TGN, Env is targeted for degradation in the lysosome instead of the assembly site of the plasma membrane. This mistrafficking of Env for degradation could be due to altered interactions with Gag, as Gag itself is mistrafficked to the assembly site in Ubc9 knockdown cells. Another potential mechanism is that as Gag is mistrafficked to the assembly site in Ubc9 knockdown cells, it may not stabilize Env at the plasma membrane causing Env to be quickly endocytosed and degraded. In Ubc9 knockdown cells, a portion of endocytosed Env may then be targeted for degradation in the lysosome instead of recycling back to the TGN. Alternatively, the trafficking or function of yet unknown cellular factor(s) that are involved in Env stability and packaging into assembling virions may have been disrupted possibly altering Gag/Ubc9 interactions (question mark).</p

Env and Gag stability in Ubc9 knockdown cells in the presence of proteasome inhibitor MG132.

<p>293T cells were transfected with Ubc9 siRNA and pNL4-3 as in previous experiments. MG132 (10µM) was added to the culture media for 1 hour prior to pulse chase experiments and was maintained throughout the experiment. Cells were pulse (P) labeled with [³⁵S] methionine/cysteine for 1 hour and then chased for 2 and 4 hours. Cell and media associated viral proteins were solublized and immunoprecipitated with pooled AIDS patient sera, split equally, and incubated for 3.5 hours at 37°C in the presence, or absence of Endoglycosidase H _f (Endo H _f). Samples were separated by SDS PAGE and visualized by phosphorimaging using The Discovery Series Quantity One software. A representative, over-exposed gel is shown so that partially Endo H_f resistant Env can be more easily visualized. Viral proteins and their positions in the gel are labeled on the left. The identity of Endo H_f, untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo H_f sensitive forms gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo H_f resistant forms of gp160 that have had their glycans modified in the TGN are labeled as gp160r. Forms of gp160 that have undergone glycan modification in the TGN but have not been cleaved are denoted as gp160* in Endo H_f untreated samples.</p