Noninfectious Particles Research Articles

Critical Review of Norovirus Surrogates in Food Safety Research: Rationale for Considering Volunteer Studies

The inability to propagate human norovirus (NoV) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses, like feline calicivirus (FCV) and murine norovirus-1 (MNV), which are propagatable in cell culture. The use of surrogates is predicated on the assumption that they generally mimic the viruses they represent; however, studies are proving this concept invalid. In direct comparisons between FCV and MNV, their susceptibility to temperatures, environmental and food processing conditions, and disinfectants are dramatically different. Differences have also been noted between the inactivation of NoV and its surrogates, thus questioning the validity of surrogates. Considerable research funding is provided globally each year to conduct surrogate studies on NoVs; however, there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies. Human challenge studies are needed to determine which processing techniques are effective in reducing NoVs in foods. A major obstacle to clinical trials on NoVs is the perception that such trials are too costly and risky, but in reality, there is far more cost and risk in allowing millions of unsuspecting consumers to contract NoV illness each year, when practical interventions are only a few volunteer studies away. A number of clinical trials have been conducted, providing important insights into NoV inactivation. A shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic, practical, and scientifically valid processing approaches to improve food safety.

The Highly Conserved Glycan at Asparagine 260 of HIV-1 gp120 Is Indispensable for Viral Entry

Carbohydrate-binding agents bind to the N-glycans of HIV-1 envelope gp120 and prevent viral entry. Carbohydrate-binding agents can select for mutant viruses with deleted envelope glycans. Not all glycosylation motifs are mutated to the same extent. Site-directed mutagenesis revealed that deletions destroying the highly conserved (260)NGS(262) glycosylation motif resulted in non-infectious virus particles. We observed a significant lower CD4 binding in the case of the N260Q mutant gp120 virus strains, caused by a strikingly lower expression of gp120 and gp41 in the virus particle. In addition, the mutant N260Q HIV-1 envelope expressed in 293T cells was unable to form syncytia in co-cultures with U87.CD4.CXCR4.CCR5 cells, due to the lower expression of envelope protein on the surface of the transfected 293T cells. The detrimental consequence of this N-glycan deletion on virus infectivity could not be compensated for by the creation of novel glycosylation sites near this amino acid, leaving this uncovered envelope epitope susceptible to neutralizing antibody binding. Thus, the Asn-260 glycan in the gp120 envelope of HIV-1 represents a hot spot for targeting suicidal drugs or antibodies in a therapeutic effort to efficiently neutralize a broad array of virus strains.

Noninfectious Retrovirus Particles Drive the Apobec3/Rfv3 Dependent Neutralizing Antibody Response

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.

HIV Gag and PR: partners in resistance to protease and maturation inhibitors

Maturation is an essential step in the HIV-1 life-cycle. It is the transition of immature, non-infectious virus particles to mature and infectious virions triggered by the proteolytic cleavage of the precursor Gag and GagPol polyproteins by the viral enzyme protease. HIV-1 protease recognizes the asymmetric shape of the peptide substrates, rather than a particular amino acid sequence. These peptides have a super-imposable secondary structure, yielding a substrate envelope which fits within the protease substrate-binding region. There are however, a few differences between the substrates in which amino acid side-chains protrude out of the envelope which contribute to the different rates of cleavage. Detailed knowledge of the structure of HIV protease and its substrate has led to the development of specific protease inhibitors (Pls), which have played a major role in antiviral therapy. They bind protease with high affinity but tend to occupy more space than the natural substrates. Resistance to PI is usually a stepwise process in which a substitution in the substrate-binding cleft of the protease is observed first. This results in an overall enlargement of the catalytic site and decreased binding to the inhibitor and, in parallel, to some decrease in binding to the natural substrate and to decreased viral replication. Several studies have identified an association between selection of protease mutations and mutations in the Gag substrate. We have shown that substitutions in the NC/p1 cleavage site alone, without any alterations in the viral protease were selected during in vitro PI exposure. These NC/ p1 changes conferred PI resistance, which could directly be related to increased gag processing. A detailed analysis of clinical isolates of patients on PI therapy also indicated that NC/p1 mutations strongly contribute to PI resistance besides compensating for a loss in viral replication. A novel class of maturation inhibitors target the gag structural proteins instead of the viral protease. Bevirimat (BVM; PA-457, MPC-4326) was the first of these maturation inhibitors to go into clinical trials. The CA/p2 cleavage site has been identified as the bevirimat target region by western-blotting and in vitro resistance selection studies. Clinical studies (phase 2b) showed that baseline polymorphisms slightly downstream of the CA/p2 cleavage site, known as the QVT-motif also confer resistance. We have shown that bevirimat resistance mutations occur more frequently in PI resistant isolates compared to isolates from PI treatment naive individuals and this could mainly be attributed to mutations in the QVT-motif. Furthermore, we have shown that PI resistance mutations alter the bevirimat resistance profiles selected in vitro and impact the level of bevirimat resistance. All these findings signify the strong interaction between the viral protease and its substrate suggesting that PI therapy can have important consequences for future treatment with maturation inhibitors.

Spontaneous immunological targeting of endogenous retrovirus K is directed against the envelope protein in rhesus macaques

Endogenous retroviruses (ERVs), remnants of ancient retroviral infections, have invaded the germ line of every vertebrate species including both non-human primates and humans. Most ERVs are functionally crippled by extensive deletions, mutations and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs retain the potential for biological activity and can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. Although ERV gene products are implicated in the etiology of disease, their impact during host infection with exogenous retroviruses such as human and simian immunodeficiency virus (HIV/ SIV) remains unknown. Here, we identify the homologue of the biologically active human ERV-K, macaque ERV-K (macERV-K), which retainsintact open reading frames in the macaque genome. Indeed, we isolated a fully-spliced mRNA product encoding macERV-K Env, which contains all the structural features of a canonical retroviral Envelope protein. Furthermore, we identified robust CD8+ and CD4+ T cell responses directed at macERV-K Env in one SIV-infected Indian rhesus macaque. The macERV-K Envspecific CD4+ T cell response in this animal was larger than the entire 5lV-specific CD4+ T cell response and had a uniform effector memory phenotype, indicating continual low-level antigen exposure. In support of this, we also identified antibody responses targeting macERV-K in this animal. Although SIV infection did not appear to trigger ERV Env-specific T cell responses, these data suggest that Env proteins from ERVs can be active during infections with exogenous retroviruses. The interaction of ERV Env proteins expressed in HIV-infected cells may have major implications for the pathogenicity of HIV and warrants further investigation.

Targeting the glycans of the HIV envelope as a therapeutic tool

Carbohydrate-binding agents (CBA) represent a broad family of agents that recognize specific glycan conformations. Several members of this compound family including peptidicprokaryotic, plant, invertebrate and vertebrate lectins, but also the well-defined non-peptidicpradimicin and benanomicin antibiotics have shown to be endowed with anti-HIV activity. These compounds inhibit entry of virus particles in susceptible T-lymphocytes and macrophages, block syncytia formation between HIV-infected and non-infected T-lymphocytes, prevent capture of HIV by DC-SIGN-expressing cells and by the macrophage mannose receptor and prevent subsequent transmission of captured HIV to susceptible T-lymphocytes. No other antiviral class of compounds have been demonstrated to block these four important ways of HIV infection/transmission, which make the CBAs an interesting new family of potential anti-HIV drugs that may be particularly useful for both systemic and topical (i.e. microbicidal) applications. CBAs bind to the glycans of the HIV envelope gpl 20 and block viral entry, most likely by freezing the envelope conformation during viral entry. Escalating drug pressure in HIV-infected cell cultures result in the selection of mutant virus strains predominantly having one or several N-glycan deletions in their envelope gp120.ln this respect, many CBAs have a high genetic barrier since several N-glycan deletions seem to be required for significant phenotypic drug resistance. Interestingly, mutant virus strains containing an increasing amount of N-glycan deletions in gp120 tend to show a compromised infectivity potential. Moreover, we have found one specific, highly conserved N-glycan in HIV-1 gp120, whose deletion results in the production of noninfectious virus particles lacking gp41gp120 in their envelope. This N-glycan may be regarded as a hot-spot for a targeted therapeutic intervention. Also, it is expected that if mutant virus particles lacking N-glycans in gp120 are selected under CBA pressure in vivo, as shown to occur in drug-exposed HIV-and SIV-infected cell cultures, previously hidden conserved immunogenic epitopes become exposed, allowing the immune system to specifically trigger an efficient immune response against these viruses. An in vivo study using SIV-infected monkeys has been planned to explore this new therapeutic concept.

Recent Developments of Peptidomimetic HIV-1 Protease Inhibitors

HIV protease plays a crucial role in the viral life cycle by processing the viral Gag and Gag-Pol polyproteins into structural and functional proteins essential for viral maturation. Inhibition of HIV-1 protease leads to the production of noninfectious virus particles and hence is an important therapeutic target for antiviral therapy in AIDS patients. Among many strategies to combat this disease, highly active antiretroviral therapy (HAART) with HIV protease inhibitors (PIs) in combination with reverse transcriptase inhibitors and fusion inhibitor continues to be the first line treatment for control of HIV infection. However, the rapid emergence of drug-resistant HIV-1 strains and the appearance of cross-resistance are severely limiting the long-term treatment options. Thus, numerous efforts have been made in the design and synthesis of novel protease inhibitors with broad-spectrum activity against multidrug-resistant HIV-1 variants by medicinal chemists. This review will focus on the substrate-based drug design of novel peptidomimetic PIs in recent years since 2006.

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO ₃⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO ₃⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV.

Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.

Virus inactivation during coagulation with aluminum coagulants

We used the bacteriophages Qβ and MS2 to determine whether viruses are inactivated by aluminum coagulants during the coagulation process. We performed batch coagulation and filtration experiments with virus-containing solutions. After filtering the supernatant of the coagulated solution through a membrane with a pore size of 50 nm, we measured the virus concentration by both the plaque forming unit (PFU) and polymerase chain reaction (PCR) methods. The virus concentration determined by the PFU method, which determines the infectious virus concentration, was always lower than that determined by the PCR-based method, which determines total virus concentration, regardless of infectivity. This discrepancy can be explained by the formation of aggregates consisting of several virus particles or by the inactivation of viruses in the coagulation process. The former possibility can be discounted because (i) aggregates of several virus particles would not pass through the 50-nm pores of the filtration membrane, and (ii) our particle size measurements revealed that the virus particles in the membrane filtrate were monodispersed. These observations clearly showed that non-infectious Qβ particles were present in the membrane filtrate after the coagulation process with aluminum coagulants. We subsequently revealed that the viruses lost their infectivity after being mixed with hydrolyzing aluminum species during the coagulation process.

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1–derived lentiviral vectors

Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.

A Single Polymorphism in HIV-1 Subtype C SP1 Is Sufficient To Confer Natural Resistance to the Maturation Inhibitor Bevirimat

3-O-(3',3'-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.

Unraveling how Rfv3/Apobec3 promotes the retrovirus-specific neutralizing antibody response (105.12)

Abstract Recovery from Friend retrovirus gene 3 (Rfv3) is a classical autosomal dominant gene that facilitates recovery from viremia and disease by promoting a more potent neutralizing antibody (NAb) response. We previously reported that Rfv3 is encoded by Apobec3, a deoxycytidine deaminase that can restrict a broad range of retroviruses. However, the mechanism for how Apobec3 promotes NAb responses and the nature of protective NAbs remain unclear. Apobec3 is related to Activation Induced Deaminase, but hapten-specific antibody affinity maturation is unaffected in Apobec3 deficient mice. In contrast, Apobec3 restriction of acute FV infection in immune cells is associated with more vigorous induction of germinal center B cells, increased B cell maturation and decreased hypergammaglobulinemia. Surprisingly, while Apobec3 reduced acute plasma infectious virus titers, plasma viral RNA loads were maintained. These plasma virions encounter an early reverse transcription block with minimum G-to-A mutations. Thus, Apobec3 appears to promote NAb responses by (1) reducing virus-induced immune dysfunction, while (2) facilitating substantial release of noninfectious viral particles, presumably to prime the B cell response. Finally, enhanced NAb responses in Rfv3/Apobec3 resistant mice correlated with virus-specific IgG1 and IgG3 titers. These findings highlight a fascinating interplay between innate Apobec3 restriction and humoral immunity, with potential implications for HIV vaccine development.

Purification and concentration of non-infectious West Nile virus-like particles and infectious virions using a pseudo-affinity Cellufine Sulfate column

Affinity column chromatography is a promising method for the purification of flavivirus particles that can supplement or potentially replace diafiltration and sucrose density centrifugation. In this study, the purification of West Nile Virus (WNV) antigens via Cellufine Sulfate column chromatography was examined. Virus-like particles (VLPs) produced by the expression of the prM and E genes were separated from most of the contaminant proteins with 0.2–0.4M NaCl, but still retained their spherical forms and immunogenicity in mice. The column, with a 1mL bed-volume, concentrated WN-VLPs a minimum of 15 fold from culture supernatants. A heparin analogue, suramin, competitively eluted WN-VLPs, but sulphated polysaccharides, such as heparin, heparin sulfate and dextran sulfate, did not. Furthermore, 2.4×109 plaque forming units of WNV and 196μg of the viral antigens were recovered from 60mL of infected culture medium at high yields (93% and 96%, respectively). These results indicate that, in addition to conventional methods, Cellufine Sulfate column chromatography is an effective preparation technique for WNV particulate antigens that does not impair the antigen virological characteristics.

Screening of fruit products for norovirus and the difficulty of interpreting positive PCR results.

Despite recent norovirus (NoV) outbreaks related to consumption of fruit products, little is known regarding the NoV load on these foods. Therefore, 75 fruit products were screened for NoV presence by using an evaluated in-house NoV detection methodology consisting of a NoV extraction method and a reverse transcription quantitative PCR assay. Additionally, the fruit samples were screened for bacterial pathogens and bacterial hygiene indicators. Results of the NoV screening showed that 18 of 75 samples tested positive for GI and/or GII NoV despite a good bacteriological quality. The recovery of murine norovirus 1 virus particles acting as process control was successful in 31 of 75 samples with a mean recovery efficiency of 11.32% ± 6.08%. The level of detected NoV genomic copies ranged between 2.5 and 5.0 log per 10 g. NoV GI and/or GII were found in 4 of 10, 7 of 30, 6 of 20, and 1 of 15 of the tested raspberries, cherry tomatoes, strawberries, and fruit salad samples, respectively. However, confirmation of the positive quantitative PCR results by sequencing genotyping regions in the NoV genome was not possible. Due to the nature of the method used (reverse transcription quantitative PCR) for detection of genomic material, no differentiation was possible between infectious and noninfectious viral particles. No NoV outbreaks related to the tested fruit product types were reported during the screening period, which hampers a conclusion as to whether these unexpected high numbers of NoV-positive results should be perceived as a public health threat. These results, however, may indicate a prior NoV contamination of the tested food samples throughout the fresh produce chain.

Phase 1 Safety and Immunogenicity Testing of DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus-like Particles

Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants. Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM. MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine.

Virus Trap in Human Serum Albumin Nanotube

Infectious hepatitis B virus (HBV), namely Dane particles (DPs), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 ± 16 nm outer diameter and 59 ± 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 × 10(7) M(-1). Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

Rhesus and Human Cytomegalovirus Glycoprotein L Are Required for Infection and Cell-to-Cell Spread of Virus but Cannot Complement Each Other

Rhesus cytomegalovirus (RhCMV), the homolog of human cytomegalovirus (HCMV), serves as a model for understanding the pathogenesis of HCMV and for developing candidate vaccines. In order to develop a replication-defective virus as a vaccine candidate, we constructed RhCMV with glycoprotein L (gL) deleted. RhCMV gL was essential for viral replication, and virus with gL deleted could only replicate in cells expressing RhCMV gL. Noncomplementing cells infected with RhCMV with gL deleted released intact, noninfectious RhCMV particles that were indistinguishable from wild-type RhCMV by electron microscopy and could be rescued by treatment of cells with polyethylene glycol. In addition, noncomplementing cells infected with RhCMV with gL deleted produced levels of gB, the major target of neutralizing antibodies, at levels similar to those observed in cells infected with wild-type RhCMV. Since RhCMV and HCMV gL share 53% amino acid identity, we determined whether the two proteins could complement the heterologous virus. Cells transfected with an HCMV bacterial artificial chromosome with gL deleted yielded virus that could replicate in human cells expressing HCMV gL. This is the second HCMV mutant with an essential glycoprotein deleted that has been complemented in cell culture. Finally, we found that HCMV gL could not complement the replication of RhCMV with gL deleted and that RhCMV gL could not complement the replication of HCMV with gL deleted. These data indicate that RhCMV and HCMV gL are both essential for replication of their corresponding viruses and, although the two gLs are highly homologous, they are unable to complement each another.

Analysis of the N-terminal region of the murine leukemia virus nucleocapsid protein

Lentiviruses such as the human immunodeficiency virus (HIV-1) and alpharetroviruses such as Rous Sarcoma virus encode an element that spans the precursor Gag (PrGag) protein capsid (CA) C-terminus, a spacer peptide (SP), and the N-terminus of nucleocapsid (NC). Perturbation of this element causes the assembly of aberrant, non-infectious virus particles. To determine whether this element is conserved in gammaretroviruses such as the Moloney murine leukemia virus (MLV), we examined the effects of insertion mutations in the N-terminal portion of the MLV NC coding region. Interestingly, we found that insertions of as many as twenty residues after the twelfth residue of MLV NC yielded proteins that directed the efficient assembly of virus particles. Virus morphologies and crosslink profiles appeared normal, and assembled viruses retained significant levels of infectivity in single cycle infection assays. Two variants were examined in the context of replicating virus constructs, and the mutations were found to be maintained during multiple rounds of infection in a cell culture system. These results suggest that the alpharetrovirus and lentivirus assembly elements either are not needed for gammaretroviruses, or are replaced by an alternative assembly element. Our results also indicate that the N-terminal region of MLV NC is amenable to genetic manipulation.