Deletion or green fluorescent protein tagging of the pUL35 capsid component of pseudorabies virus impairs virus replication in cell culture and neuroinvasion in mice

Abstract

To facilitate tracing of virion movement, the non-essential capsid proteins pUL35 of herpes simplex virus type 1 and pseudorabies virus (PrV) have been tagged with green fluorescent protein (GFP). However, the biological relevance of PrV pUL35 and the functionality of the fusion proteins have not yet been investigated in detail. We generated PrV mutants either lacking the 12 kDa UL35 gene product, or expressing GFP fused to the N terminus of pUL35. Remarkably, both mutants exhibited significant replication defects in rabbit kidney cells, which could be corrected in pUL35-expressing cells. After intranasal infection of mice both mutants showed delayed neuroinvasion, and survival times of the animals were extended to 3 days, compared with 2 days after wild-type infection. Thus, fusion of pUL35 with GFP resulted in a non-functional protein, which has to be considered for the use of corresponding mutants in tracing studies.

Translocation of Incoming Pseudorabies Virus Capsids to the Cell Nucleus Is Delayed in the Absence of Tegument Protein pUL37

“…Although eGFP fusion can result in loss of pUL35 function (27), it has been shown for HSV-1 that dynein-mediated transport is not impaired, even by complete loss of pUL35 (1,10). Genome analyses of the obtained recombinant PrV-⌬UL37/UL35GFP confirmed the expected deletion of UL37 codons 15 to 778 (of 920) (25), as well as in-frame fusion of the eGFP and pUL35 coding sequences (results not shown).…”

“…To analyze the role of pUL37 in early events of herpesvirus infection, we constructed and characterized a novel pUL37 deletion mutant of PrV based on wild-type PrV strain Kaplan (PrV-Ka) (22), which expresses a fusion protein of the nonessential small capsid protein pUL35 with enhanced green fluorescent protein (eGFP) (4,5,27). Although eGFP fusion can result in loss of pUL35 function (27), it has been shown for HSV-1 that dynein-mediated transport is not impaired, even by complete loss of pUL35 (1,10).…”

“…Genome analyses of the obtained recombinant PrV-⌬UL37/UL35GFP confirmed the expected deletion of UL37 codons 15 to 778 (of 920) (25), as well as in-frame fusion of the eGFP and pUL35 coding sequences (results not shown). For protein characterization, RK13 or complementing RK13-UL37 cells (25) were infected with PrV-⌬UL37/UL35GFP, PrV-UL35GFP (27), PrV-⌬UL37 (25), or PrV-Ka at a multiplicity of infection (MOI) of 1 for 30 h, and infected cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analyses were performed as described with monospecific anti-pUL35 (27) or anti-pUL37 rabbit sera (25) (Fig.…”

“…For protein characterization, RK13 or complementing RK13-UL37 cells (25) were infected with PrV-⌬UL37/UL35GFP, PrV-UL35GFP (27), PrV-⌬UL37 (25), or PrV-Ka at a multiplicity of infection (MOI) of 1 for 30 h, and infected cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analyses were performed as described with monospecific anti-pUL35 (27) or anti-pUL37 rabbit sera (25) (Fig. 1).…”

Molecular cloning and characterization of the pseudorabies virus US1 gene

“…During the lytic cycle of PRV infection, viral genes are expressed in a cascade of 3 temporally distinct and functionally interdependent phases termed immediate-early (IE), early (E), and late (L). Although several PRV genes, including the unique IE gene IE180 (Tomioka et al, 2008), 3 E genes EP0 (Brukman and Enquist, 2006), UL54 (Li et al, 2011a,b), and UL23 (Ferrari et al, 2000), and many L genes such as capsid genes UL25 (Coller et al, 2007) and UL35 (Krautwald et al, 2008), tegument genes UL36, UL37 (Luxton et al, 2006), and UL41 (Lin et al, 2010), and envelope genes UL27 (Nixdorf et al, 2001a), UL44 (Kramer et al, 2011), US8, and UL10 (Nixdorf et al, 2001b), have been extensively studied, the function of US1 and its protein product ICP22 (PICP22) is less well understood.…”