Ubiquitin-dependent virus particle budding without viral protein ubiquitination

An essential step in the release of an extracellular enveloped virus particle is a budding event that ultimately separates virion and host cell membranes. For many enveloped viruses, membrane fission requires the recruitment of the class E vacuolar protein sorting (VPS) machinery by short, virally encoded peptide sequences termed "late-budding" or "L" domains. Some L-domain peptide sequences (e.g., PSAP) bind directly to components of class E VPS machinery, whereas others (e.g., PPxY) access it indirectly by recruiting ubiquitin ligases. Additionally, ubiquitin itself is known to be generally important for the fission of virion from cellular membranes, and because ubiquitination of cellular transmembrane proteins can signal the recruitment of class E machinery, a popular model is that deposition of ubiquitin on viral structural proteins mediates class E machinery recruitment. To test this model, we took advantage of a retroviral Gag protein from the prototypic foamy virus (PFV) that is almost devoid of ubiquitin acceptors, and we engineered it to generate extracellular virus-like particles in the complete absence of other viral proteins. Notably, we found that particle budding, induced by a class E VPS machinery-binding L domain (PSAP), proceeded efficiently in the absence of ubiquitin acceptors in PFV Gag. Moreover, when particle release was engineered to be dependent on a viral PPXY motif, the requirement for a catalytically active ubiquitin ligase was maintained, irrespective of the presence or absence of ubiquitin acceptor sites in PFV Gag. Thus, in this model system, ubiquitin conjugation to transacting factors, not viral proteins, appears critical for ubiquitin-dependent enveloped viral particle release.     

Endogenous neosynthesis vs. cross-presentation of viral antigens for cytotoxic T cell priming

Induction of antiviral cytotoxic T lymphocytes (CTLs) has been proposed to require cross-presentation of viral antigens derived from infected extralymphatic host cells by antigen-presenting cells (APC). This postulated mechanism of cross-priming is thought to be essential for CTL responses against viruses that do not infect professional APC, e.g., because of absence of the specific virus receptor. Here, we show for the human pathogen poliovirus that naturally nonpermissive murine APC acquire viral RNA in vivo independently of the cellular virus receptor. Uptake of poliovirus or polioviral RNA initiated neosynthesis of viral antigen to an extent sufficient to prime CTLs in vivo, which were detectable 2-3 wk after infection. Our results do not only indicate that experiments studying cross-presentation and cross-priming by using potentially amplifiable or translatable materials need careful examination, but they also question the general biological importance of cross-presentation and cross-priming in antiviral CTL responses.     

Viral persistence in vivo through selection of neutralizing antibody-escape variants

Despite initial virus control by CD8(+) cytotoxic T lymphocytes (CTLs), noncytopathic or variably cytopathic viruses (e.g., hepatitis B and C viruses, HIV) are able to establish persistent infections. The role of neutralizing antibodies (nAbs) in controlling disease progression is unclear. Therefore, the phenomenon of viral evasion from the nAb response and its implications for virus persistence remain controversial. Here we demonstrate nAb-mediated viral clearance in CTL-deficient mice infected with the prototypic noncytopathic lymphocytic choriomeningitis virus (strain WE). During prolonged CTL absence, neutralization-resistant virus mutants were selected in individual mice within 70-90 days. In naive animals infected with these virus variants only low nAb responses were induced, resulting in an increased tendency of virus to persist.     

Activation of the Mason–Pfizer monkey virus protease within immature capsids in vitro

For all retroviruses, the completion of the viral budding process correlates with the activation of the viral protease by an unknown mechanism, and, as the structural (Gag) polyproteins are cleaved by the viral protease, maturation of the immature virus-like particle into an infectious virion. Unlike most retroviruses, the Mason-Pfizer monkey virus Gag polyproteins assemble into immature capsids within the cytoplasm of the cell before the viral budding event. The results reported here describe a unique experimental system in which Mason-Pfizer monkey virus immature capsids are removed from the cell, and the protease is activated in vitro by the addition of a reducing agent. The cleavage of the protease from the precursor form is a primary event, which proceeds with a half time of 14 min, and is followed by authentic processing of the Gag polyproteins. Activity of the viral protease in vitro depends on pH, with an increase in catalytic rates at acidic and neutral pH. The initiation of protease activity within immature capsids in vitro demonstrates that viral protease activity is sensitive to oxidation-reduction conditions, and that the viral protease can be activated in the absence of viral budding.     

Approaches to the diagnosis of viral pneumonias in the immunocompromised host: the importance of assaying cytopathogenic viral effects in bronchoalveolar lavage cells

Pneumonopathic conditions in the immunocompromised host (IH) are frequent and often serious. Rapid diagnosis is essential and is made possible by bronchoalveolar lavage (BAL). Sixty-two pneumonopathic episodes in 53 immunocompromised patients were examined by BAL, for viral cytopathogenic effects (CPE) in isolated cells, with appropriate viral culture techniques. Viral culture was positive in seventeen of the eighteen episodes in renal allograft recipients and AIDS patients as against eight of the fourty-four episodes in other causes of IH (p less than 0.001). CPE was found thirteen times; in seven cases it was characteristic of cytomegalovirus. Positive viral culture and CPE were shown simultaneously during thirteen episodes in eleven patients. Ten patients died (autopsies performed in three cases confirmed viral presence). Positive viral culture with absence of CPE was observed in twelve cases. There were only four fatalities in this group (the autopsies performed in three cases did not establish the presence of a virus in the pulmonary parenchyma). The percentage of lymphocytes was high in both groups of patients (18.6 +/- 2.8%). CPE is a simple and rapid examination for the diagnosis of viral pneumonopathology in the IH. Prognosis at present is gloomy; more complex examinations such as viral cultures and/or identification of the virus by immunofluorescence will be indicated only when effective antiviral agents become available.     

Virome Variation during Sea Star Wasting Disease Progression in Pisaster ochraceus (Asteroidea,Echinodermata)

Sea star wasting disease (SSWD) is a condition that has affected asteroids for over 120 years, yet mechanistic understanding of this wasting etiology remains elusive. We investigated temporal virome variation in two Pisaster ochraceus specimens that wasted in the absence of external stimuli and two specimens that did not experience SSWD for the duration of our study, and compared viromes of wasting lesion margin tissues to both artificial scar margins and grossly normal tissues over time. Global assembly of all SSWD-affected tissue libraries resulted in 24 viral genome fragments represented in >1 library. Genome fragments mostly matched densoviruses and picornaviruses with fewer matching nodaviruses, and a sobemovirus. Picornavirus-like and densovirus-like genome fragments were most similar to viral genomes recovered in metagenomic study of other marine invertebrates. Read recruitment revealed only two picornavirus-like genome fragments that recruited from only SSWD-affected specimens, but neither was unique to wasting lesions. Wasting lesion margin reads recruited to a greater number of viral genotypes (i.e., richness) than did either scar tissue and grossly normal tissue reads. Taken together, these data suggest that no single viral genome fragment was associated with SSWD. Rather, wasting lesion margins may generally support viral proliferation.     

Proteasome-dependent,ubiquitin-independent degradation of the Rb family of tumor suppressors by the human cytomegalovirus pp71 protein

Most of the substrates degraded by the proteasome are marked with polyubiquitin chains. However, there are a limited number of examples of nonubiquitinated proteins that are degraded by the proteasome. Here, we describe the degradation of the retinoblastoma family of tumor suppressor proteins by the proteasome in the absence of polyubiquitination. The retinoblastoma protein (p105), p107, and p130 are each targeted for degradation by the pp71 protein, which is encoded by the UL82 gene of human cytomegalovirus. It functions to direct their degradation in the absence of other viral proteins. While the pp71-mediated degradation of the retinoblastoma family of proteins requires proteasome function, it occurs without the attachment of ubiquitin to the substrates and in the absence of a functioning ubiquitin-conjugation system.     

Casein kinase II specifically nucleotidylylates in vitro the amino acid sequence of the protein encoded by the alpha 22 gene of herpes simplex virus 1.

An earlier report has shown that eight viral proteins with a common amino acid sequence (R/P)RA(P/S)R are nucleotidylyated in vitro by nuclear extracts from cells infected with herpes simplex virus 1. One, the product of the alpha 22 gene, is nucleotidylylated in the absence of viral proteins made late in infection. A chimeric protein (GST22P) consisting of amino acids 50-200 of the alpha 22 coding sequence fused to the C terminus of the glutathione S-transferase was nucleotidylylated by enzymes in nuclear extracts of infected or mock-infected cells and also by a casein kinase II enzyme purified from the sea star. The enzyme did not nucleotidylylate common casein kinase II substrates (casein, phosvitin) and the reaction was inhibited by heparin. The results are consistent with the hypothesis that nucleotidylylation of the eight viral proteins involves casein kinase II.     

Neuronal survival strategies in the face of RNA viral infection

Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, viral infections that result in neuronal depletion, either by viral lysis or by induction of the cytolytic immune response, would likely lead to profound neurologic impairment. However, many viral infections that result in tissue destruction elsewhere in the host produce few overt symptoms in the CNS, despite readily detectable virus expression. This observation has lead to the speculation that neurons possess strategies to limit the replication and spread of otherwise cytopathic viruses. These strategies either favor the clearance of virus in the absence of appreciable neuronal loss or promote the establishment of noncytolytic persistent infections. This review discusses some of these strategies, with an emphasis on how such survival techniques lessen the potential for CNS neuropathology.     

Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus.

The genome of human immunodeficiency virus encodes a protein that dramatically elevates amounts of viral proteins. The precise mechanism of this trans-activation remains to be established. It has been reported that trans-activation can occur without major changes in the levels of mRNA. We constructed recombinant plasmids containing those viral sequences required in cis for trans-activation linked to the chloramphenicol acetyltransferase gene. These plasmids were introduced into cultured cells in either the presence or absence of a second plasmid that directed expression of the viral trans-activator protein. Expression of the chloramphenicol acetyltransferase gene was measured at the level of protein (by enzymatic assay) and RNA (by ribonuclease protection and primer extension). Our results demonstrate that trans-activation is accompanied by large increases in mRNA levels; these increases may be sufficient to explain the elevated levels of trans-activated protein.     

Inactivation of a European strain of West Nile virus in single- donor platelet concentrate using the INTERCEPT blood system

BACKGROUND AND OBJECTIVE: In order to prevent West Nile virus (WNV) contaminations by transfusion, the French National Blood Service decided to evaluate the INTERCEPT Blood System's efficiency on a European strain. MATERIALS AND METHODS: Culture supernatant of WNV was used to infect six platelets concentrates. Viral titre was determined by plaque reduction neutralization test before and after viral inactivation using the INTERCEPT Blood System. RESULTS: In all assays, the absence of plaque forming unit was observed after viral inactivation. The log reduction observed ranged between > 5.1 logs to > 5.2 logs. CONCLUSION: INTERCEPT Blood System is a commercially viral inactivation method potentially useful in order to prevent WNV transmission by blood products in France during re-emerging outbreaks.     

Endogenous oncornaviral antigen in the bursa of Fabricius of 15B X 7(2) chickens.

Oncornaviral antigen was detected in the bursal epithelium and in a subpopulation of bursal follicular cells of 15B X 72 chickens. This antigen is present in the bursal epithelium at 11 days of embryogenesis and persists there for at least 3 weeks after hatching. The absence of detectable antigen in the intestinal epithelium contiguous to the bursal epithelium indicates that the accumulation of viral antigen is a specific property of the bursal epithelium. The observation of C-type particles in the intraepithelial spaces suggests that the viral antigen in synthesized and assembled into virions by the bursal epithelial cells. In embryonic bursas, viral antigen-positve cells radiate from the surface epithelium toward the central region of the follicles. In bursas from post-hatch chickens, viral antigen-positive cells, including intrafollicular epithelial cells and cells resembling lymphocytes, are confined to the medullary region of the follicles.     

An MHV-68 Mutator Phenotype Mutant Virus,Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene,Shows Reduced Viral Fitness

Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383S^GCV, Q827R^HPMP-5azaC, G302W^PFA, K442T^PFA, G302W+K442T^PFA, C297W^HPMPO-DAPy and C981Y^CDV. Without antiviral pressure, viral clones Q827R^HPMP-5azaC, G302W^PFA, K442T^PFA and G302W+K442T^PFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383S^GCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.     

Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle.

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.     

Memory CD8+ T cells are gatekeepers of the lymph node draining the site of viral infection

It is uncertain how immunity protects against systemic viral diseases. Here, we demonstrate that in the absence of persistent virus, not only antibodies but also recall responses by long-lived memory CD8(+) T cells prevent mousepox, a disease caused by ectromelia virus, a close relative of the virus of human smallpox. Moreover, we show that to protect, recall CD8(+) T cells directly kill targets in the lymph node draining the primary site of infection thus curbing systemic viral spread. Therefore, our work provides the basis for a model where lymph nodes are not just organs where lymphocytes become activated and proliferate but also the sites where a major fight against virus spread takes place.