Asit K. Pattnaik

Essential Role for the dsRNA-Dependent Protein Kinase PKR in Innate Immunity to Viral Infection

The double-stranded (ds) RNA-dependent protein kinase PKR is considered to play an important role in interferons (IFNs) response to viral infection. Here, we demonstrate that mice lacking PKR are predisposed to lethal intranasal infection by the usually innocuous vesicular stomatitis virus, and also display increased susceptibility to influenza virus infection. Our data indicate that in normal cells, PKR primarily prevents virus replication by inhibiting the translation of viral mRNAs through phosphorylation of eIF2alpha, while concomitantly assisting in the production of autocrine IFN and the establishment of an antiviral state. These results show that PKR is an essential component of innate immunity that acts early in host defense prior to the onset of IFN counteraction and the acquired immune response.

Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1β Modulates Host Innate Immune Response by Antagonizing IRF3 Activation

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1α and NSP1β (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1β was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-κB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1β. Moreover, when tested in a porcine myelomonocytic cell line, NSP1β inhibited Sendai virus-mediated activation of porcine IFN-β promoter activity. We propose that this NSP1β-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell.

A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

Abstract The nucleotide sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Transfection of MARC-145 cells with capped in vitro transcripts derived from a full-length cDNA clone of the viral genome resulted in infectious PRRSV with growth characteristics similar to that of the parental virus. Primer extension analysis revealed that during replication, the viral polymerase corrected the two nonviral guanosine residues present at the 5′ terminus of the transfected transcripts. Animal studies showed that the cloned virus induced hyperthermia, persistent viremia, and antibody response, similar to that observed with the parental virus. Contact transmission occurred rapidly within 3 days of introduction of naı̈ve pigs into the group of clone virus-inoculated pigs. These results suggest that the cloned virus retains the in vivo virulence and contagion properties of the parental virus, thus, providing the background for reverse genetics manipulation in systematic examination of attenuation and virulence phenotypes.

Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

ABSTRACT The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3

ABSTRACT Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.

The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles

Abstract Infectious defective interfering (DI) particles of the negative-stranded RNA virus vesicular stomatitis virus (VSV) havebeen recovered from negative-sense transcripts of a plasmid that contains a full-length cDNA derived from the DI-T particle genome. In order to determine the cis-acting sequences necessary for RNA replication, encapsidation, and budding and to approximate the minimal size of RNA that can be packaged into infectious particles, we constructed a series of internal deletions in the DI cDNA to generate plasmids that could be transcribed to yield RNAs which ranged in size from 2209 nucleotides down to 102 nucleotides. All the deletion plasmids retained at least 36 nucleotides from the 5′-terminus and 51 nucleotides from the 3′-terminus of the DI genome. In cells expressing the five VSV proteins, the deleted DI RNAs were examined for their ability to be encapsidated, to replicate, and to bud to produce infectious DI particles. An RNA as small as 191 nucleotides, which contained 46 nucleotides from the 5′-end and 145 nucleotides from the 3′-end of the DI genome was encapsidated, replicated, and budded at least as efficiently as the full-length wild-type DI RNA. In contrast, a 102-nucleotide RNA that contained only the 51 nucleotides from the 5′-end of the DI RNA and its perfect 51-nucleotide complement at the 3′-end replicated poorly and failed to bud infectious DI particles. However, an RNA with an insertion of 1499-nucleotide “stuffer” sequences of non-VSV origin between the two 51-nucleotide complementary termini not only replicated but also budded infectious particles. These data show that the signals necessary for RNA encapsidation, replication, and packaging into infectious DI particles are contained within the 5′-terminal 36 nucleotides and the 3′-terminal 51 nucleotides of the DI RNA genome. Furthermore, the results show that a heterologous sequence can be replicated and packaged into infectious particles if it is flanked by the DI RNA termini.

Identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones

In order to determine virulence associated genes in porcine reproductive and respiratory syndrome virus (PRRSV), a series of chimeric viruses were generated where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain (Prime Pac). Initial genome-wide scanning using a sow reproductive failure model indicated that non-structural (ORF 1a and 1b) and structural (ORF2-7) genomic regions appear to be sites where virulence determinants of PRRSV may reside. These results thus confirm the multigenic character of PRRSV virulence. Additional chimeras containing each individual structural ORFs (2 through 7) of Prime Pac and ORF5 of Neb-1 (parental strain of Prime Pac) within the FL12 backbone were generated and tested individually for further mapping of virulence determinants. Our results allow to conclude that NSP3-8 and ORF5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1-3, NSP10-12 and ORF2.

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.