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The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity. Author summary: Viral glycoproteins are critical determinants of host cell tropism, immunity, and pathogenesis. Vaccinia virus was used for the most successful immunization program in history, and poxviruses continue to be used as vaccine vectors. Here, we report that vaccinia virus extracellular virion (EV) protein F13 plays an important, previously unappreciated, role in controlling glycoprotein incorporation, and that there is a direct relationship between glycoprotein concentrations and subsequent infectivity. Crucially, manipulation of the EV glycoprotein concentrations altered pathogenesis and lethality in an in vivo infection model, but did not markedly alter the induced immune response. These results have important implications that inform the design of safer and more efficacious poxvirus-based vaccine vectors by altering glycoprotein content. [ABSTRACT FROM AUTHOR]

Orthopoxviruses produce two antigenically distinct infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane are a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV and which plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV-specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope-tagged version of MC021L. The resulting virus (vMC021L-HA) had a small-plaque phenotype compared to vF13L-HA but larger than vF13L. The localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced amounts of EV similar to those produced by vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding and an increase in dissolution, both of which correlated with a small-plaque phenotype. [ABSTRACT FROM AUTHOR]

The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution. IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus. [ABSTRACT FROM AUTHOR]

Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2–3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host’s ability to eliminate HIV. [ABSTRACT FROM AUTHOR]

The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4-IgD-memory B cell population, the 9G4+IgD-memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward ''IgM only'' memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies. [ABSTRACT FROM AUTHOR]

Many enveloped RNA viruses utilize lipid rafts for the assembly of progeny virions, but the role of cholesterol, a major component of rafts, on paramyxovirus budding and virion formation is controversial. In this study, we analyzed the effects of FDA-approved cholesterol-reducing agents, gemfibrozil and lovastatin, on raft formation and assembly of human parainfluenza virus type 1 (hPIV1) and Sendai virus (SeV). Treatment of the human airway epithelial A549 cells with the agents, especially when combined, significantly decreased production of infectious hPIV1 and SeV. Mechanistic analysis indicated that depletion of cellular cholesterol reduced cell surface accumulation of envelope glycoproteins and association of viral matrix and nucleocapsids with raft membrane, which resulted in impaired virus budding and release from the cells. These results indicate that cellular cholesterol is required for assembly and formation of type 1 parainfluenza viruses and suggest that cholesterol could be an attractive target for antiviral agents against hPIV1. [ABSTRACT FROM AUTHOR]